Streptomyces avermitilis regulatory genes for increased avermectin production

ABSTRACT

The present invention is directed to compositions and methods for producing avermectins, and is primarily in the field of animal health. The present invention relates to the identification and characterization of two novel genes, herein referred to as the aveR1 and aveR2 genes, that are involved in regulating avermectin polyketide synthase (PKS) expression and avermectin biosynthesis in  Streptomyces avermitilis . The present invention is based on the discovery that inactivation of these genes results in an increase in the amount of avermectin produced by  S. avermitilis.

This application claims priority from U.S. provisional application Ser.No. 60/100,134 filed Sep. 14, 1998.

1. FIELD OF THE INVENTION

The present invention is directed to compositions and methods forproducing avermectins, and is primarily in the field of animal health.More particularly, the present invention relates to the identificationand characterization of two novel genes, herein referred to as the aveR1and aveR2 genes, that are involved in regulating avermectin polyketidesynthase (PKS) expression and avermectin biosynthesis in Streptomycesavermitilis. The present invention is based on the discovery thatinactivation of these genes results in an increase in the amount ofavermectin produced by S. avermitilis.

2. BACKGROUND OF THE INVENTION

Streptomyces species produce a wide variety of secondary metabolites,including the avermectins, which comprise a series of eight relatedsixteen-membered macrocyclic lactones with potent anthelmintic andinsecticidal activity. The eight distinct but closely related compoundsare referred to as A1a, A1b, A2a, A2b, B1a, B1b, B2a, and B2b. The “a”series of compounds refers to the natural avermectin wherein thesubstituent at the C25 position is (S)sec-butyl, and the “b” seriesrefers to those wherein the substituent at the C25 position isisopropyl. The designations “A” and “B” refer to avermectins wherein thesubstituent at C5 is methoxy and hydroxy, respectively. The numeral “1”refers to avermectins wherein a double bond is present at the C22, 23position, and the numeral “2” refers to avermectins having a hydrogen atthe C22 position and a hydroxy at the C23 position. Among the relatedavermectins, the B1 type of avermectin is recognized as having the mosteffective antiparasitc and pesticidal activity, and is therefore themost commercially desirable avermectin.

The avermectins and their production by aerobic fermentation of strainsof S. avermitilis are described, among other places, in U.S. Pat. Nos.4,310,519 and 4,429,042.

The avermectin (ave) genes, like many genes involved in the productionof secondary metabolites and other Streptomyces antibiotics, are foundclustered together on the bacterial chromosome. The ave gene cluster foravermectin biosynthesis spans a 95 kb genomic fragment of DNA whichincludes DNA encoding the avermectin polyketide synthase (PKS) (MacNeilet al., 1992, Gene 115:119-125).

The regulation of antibiotic biosynthesis in Streptomyces is perhapsbest characterized in the species Streptomyces coelicolor. Fourantibiotics produced by S. coelicolor include actinorhodin (Act),undecylprodigiosin (Red), calcium-dependent antibiotic (CDA), andmethylenomycin (Mmy). Each of these antibiotics is encoded by adifferent cluster of genetically distinct genes. Genes have beenidentified that are linked to either the Act gene cluster or the Redgene cluster that encode products which specifically regulate theexpression of the Act biosynthetic gene cluster or the Red biosyntheticgene cluster, respectively. A number of loci containing genes thatglobally regulate more than one of the antibiotic biosynthetic geneclusters have also been identified. For example, mutations in twoindependent loci, absA and absB, have been shown to block the synthesisof all four antibiotics in S. coelicolor (Brian et al., 1996, J. Bact.178:3221-3231). The absA locus has been cloned and characterized, andits gene products have been shown to be involved in a signaltransduction pathway which normally acts as a global negative regulatorof antibiotic synthesis in S. coelicolor (Brian et al., 1996, above).

U.S. Pat. No. 5,876,987 to Champness et al. relates to hyperproductionof antibiotic in Streptomyces spp. as a result of interruption of theabsA locus.

U.S. Pat. No. 5,707,839 to Denoya, and U.S. Pat. No. 5,728,561 to Denoyaet al. relate to DNA sequences encoding branched-chain alpha-ketoaciddehydrogenase complexes of Streptomyces and methods for enhancing theproduction of novel avermectins.

Understanding the mechanism by which Type I polyketide synthaseexpression is regulated in S. avermitilis will permit geneticmanipulation of the ave genes to increase the production of avermectins.

3. SUMMARY OF THE INVENTION

The present invention provides an isolated polynucleotide moleculecomprising a nucleotide sequence encoding an aveR1 gene product from S.avermitilis. In a preferred embodiment, the aveR1 gene product comprisesthe amino acid sequence of SEQ ID NO:2. In a non-limiting embodiment,the isolated polynucleotide molecule of the present invention comprisesthe nucleotide sequence of the aveR1 ORF of S. avermitilis as shown inSEQ ID NO:1 from about nt 1112 to about nt 2317. In a furthernon-limiting embodiment, the isolated polynucleotide molecule of thepresent invention comprises the nucleotide sequence of SEQ ID NO:1.

The present invention further provides an isolated polynucleotidemolecule that is homologous to a polynucleotide molecule comprising thenucleotide sequence of the aveR1 ORF of S. avermitilis as shown in SEQID NO:1 from about nt 1112 to about nt 2317.

The present invention further provides an isolated polynucleotidemolecule comprising a nucleotide sequence that encodes a polypeptidehaving an amino acid sequence that is homologous to the amino acidsequence of SEQ ID NO:2.

The present invention further provides an isolated polynucleotidemolecule consisting of a nucleotide sequence that is a substantialportion of any of the aforementioned aveR1-related polynucleotidemolecules of the present invention. In a preferred embodiment, thesubstantial portion of the aveR1-related polynucleotide moleculeconsists of a nucleotide sequence that encodes a peptide fragment of aS. avermitilis aveR1 gene product or aveR1-related homologouspolypeptide of the present invention. In a specific though non-limitingembodiment, the present invention provides a polynucleotide moleculeconsisting of a nucleotide sequence encoding a peptide fragmentconsisting of a sub-sequence of the amino acid sequence of SEQ ID NO:2.

The present invention further provides an isolated polynucleotidemolecule comprising one or more nucleotide sequences that naturallyflank the aveR1 ORF of S. avermitilis in situ. Such flanking sequencescan be selected from the nucleotide sequence of SEQ ID NO:1 from aboutnt 1 to about nt 1111, and from about nt 2318 to about nt 5045. Thepresent invention further provides an isolated polynucleotide moleculecomprising one or more nucleotide sequences that are homologous tonucleotide sequences that naturally flank the aveR1 ORF of S.avermitilis in situ. Each flanking sequence, or homolog thereof, in theisolated polynucleotide molecule of the present invention is preferablyat least about 200 nt in length. In a non-limiting embodiment, thepresent invention provides an isolated polynucleotide moleculecomprising one or more of the aforementioned nucleotide sequences thatnaturally flank the aveR1 ORF of S. avermitilis in situ, or that arehomologous to such nucleotide sequences, and further comprising one ofthe aforementioned aveR1-related nucleotide sequences of the presentinvention such as, e.g., the nucleotide sequence of the aveR1 ORF of S.avermitilis as shown in SEQ ID NO:1 from about nt 1112 to about nt 2317or substantial portion thereof.

The present invention further provides an isolated polynucleotidemolecule comprising a nucleotide sequence encoding an aveR2 gene productfrom S. avermitilis. In a preferred embodiment, the aveR2 gene productcomprises the amino acid sequence of SEQ ID NO:4. In a non-limitingembodiment, the isolated polynucleotide molecule of the presentinvention comprises the nucleotide sequence of the aveR2 ORF of S.avermitilis as shown in SEQ ID NO:3 (note: SEQ ID NO:3 is identical toSEQ ID NO:1) from about nt 2314 to about nt 3021. In a furthernon-limiting embodiment, the isolated polynucleotide molecule of thepresent invention comprises the nucleotide sequence of SEQ ID NO:3.

The present invention further provides an isolated polynucleotidemolecule that is homologous to a polynucleotide molecule comprising thenucleotide sequence of the aveR2 ORF of S. avermitilis as shown in SEQID NO:3 from about nt 2314 to about nt 3021.

The present invention further provides an isolated polynucleotidemolecule comprising a nucleotide sequence that encodes a polypeptidehaving an amino acid sequence that is homologous to the amino acidsequence of SEQ ID NO:4.

The present invention further provides an isolated polynucleotidemolecule consisting of a nucleotide sequence that is a substantialportion of any of the aforementioned aveR2related polynucleotidemolecules of the present invention. In a preferred embodiment, thesubstantial portion of the aveR2-related polynucleotide moleculeconsists of a nucleotide sequence that encodes a peptide fragment of aS. avermitilis aveR2 gene product or aveR2related homologous polypeptideof the present invention. In a specific though non-limiting embodiment,the present invention provides a polynucleotide molecule consisting of anucleotide sequence encoding a peptide fragment consisting of asub-sequence of the amino acid sequence of SEQ ID NO:4.

The present invention further provides an isolated polynucleotidemolecule comprising one or more nucleotide sequences that naturallyflank the aveR2 ORF of S. avermitilis in situ. Such flanking sequencescan be selected from the nucleotide sequence of SEQ ID NO:3 from aboutnt 1 to about nt 2313, and from about nt 3022 to about nt 5045. Thepresent invention further provides an isolated polynucleotide moleculecomprising one or more nucleotide sequences that are homologous tonucleotide sequences that naturally flank the aveR2 ORF of S.avermitilis in situ. Each flanking sequence in the isolatedpolynucleotide molecule of the present invention is preferably at leastabout 200 nt length. In a non-limiting embodiment, the present inventionprovides an isolated polynucleotide molecule comprising one or more ofthe aforementioned nucleotide sequences that naturally flank the aveR2ORF of S. avermitilis in situ, or that are homologous to such nucleotidesequences, and further comprising one of the aforementionedaveR2-related nucleotide sequences of the present invention such as,e.g., the nucleotide sequence of the aveR2 ORF as shown in SEQ ID NO:3from about nt 2314 to about nt 3021 or a substantial portion thereof.

The present invention further provides an isolated polynucleotidemolecule comprising a nucleotide sequence encoding both the aveR1 andaveR2 gene products from S. avermitilis. In a preferred embodiment, theaveR1 and aveR2 gene products comprise the amino acid sequences of SEQID NO:2 and SEQ ID NO:4, respectively. In a non-limiting embodiment, theisolated polynucleotide molecule comprises the nucleotide sequence ofthe aveR1 ORF of S. avermitilis as shown in SEQ ID NO:1 from about nt1112 to about nt 2317 and the aveR2 ORF of S. avermitilis as shown inSEQ ID NO:1 from about nt 2314 to about nt 3021. In a furthernon-limiting embodiment, the isolated polynucleotide molecule comprisesthe nucleotide sequence of SEQ ID NO:1 from about nt 1112 to about nt3021. In a further non-limiting embodiment, the isolated polynucleotidemolecule comprises the nucleotide sequence of SEQ ID NO:1.

The present invention further provides an isolated polynucleotidemolecule that is homologous to a polynucleotide molecule comprising thenucleotide sequence of both the aveR1 and aveR2 ORFs of S. avermitilis.In a non-limiting embodiment, the present invention provides an isolatedpolynucleotide molecule that is homologous to a polynucleotide moleculecomprising the nucleotide sequence of the aveR1 ORF of S. avermitilis asshown in SEQ ID NO:1 from about nt 1112 to about nt 2317, and the aveR2ORF of S. avermitilis as shown in SEQ ID NO:1 from about nt 2314 toabout nt 3021.

The present invention further provides an isolated polynucleotidemolecule comprising a nucleotide sequence that encodes a firstpolypeptide having an amino acid sequence that is homologous to theamino acid sequence of SEQ ID NO:2 and a second polypeptide having anamino acid sequence that is homologous to the amino acid sequence of SEQID NO:4.

The present invention further provides an isolated polynucleotidemolecule consisting of a nucleotide sequence that is a substantialportion of any of the aforementioned polynucleotide molecules whichcomprise a nucleotide sequence encoding both the aveR1 and aveR2 geneproducts from S. avermitilis or any of the aforementioned polynucleotidemolecules that are homologous thereto. In a specific though non-limitingembodiment, the substantial portion of the polynucleotide moleculeconsists of the nucleotide sequence of the aveR1 ORF as shown in SEQ IDNO:1 from about nt 1112 to about nt 2317. In another specific thoughnon-limiting embodiment, the substantial portion of the polynucleotidemolecule consists of the nucleotide sequence of the aveR2 ORF as shownin SEQ ID NO:3 from about nt 2314 to about nt 3021.

The present invention further provides an isolated polynucleotidemolecule comprising one or more nucleotide sequences that naturallyflank the aveR1 and aveR2 ORFs of S. avermitilis in situ. Such flankingsequences can be selected from the nucleotide sequence of SEQ ID NO:1from about nt 1 to about nt 1111, and from about nt 3022 to about nt5045. The present invention further provides an isolated polynucleotidemolecule comprising one or more nucleotide sequences that are homologousto nucleotide sequences that naturally flank the aveR1 and aveR2 ORFs ofS. avermitilis in situ. Each flanking sequence, or homolog thereof, inthe isolated polynucleotide molecule of the present invention ispreferably at least about 200 nt in length. In a non-limitingembodiment, the present invention provides an isolated polynucleotidemolecule comprising one or more of the aforementioned nucleotidesequences that naturally flank the aveR1 and aveR2 ORFs of S.avermitilis in situ, or that are homologous to such nucleotidesequences, and further comprising one of the aforementioned nucleotidesequences of the present invention that encode either or both of theaveR1 and aveR2 gene products from S. avermitilis, such as, e.g., thenucleotide sequence of the aveR1 ORF of S. avermitilis as shown in SEQID NO:1 from about nt 1112 to about nt 2317, and the nucleotide sequenceof the aveR2 ORF of S. avermitilis as shown in SEQ ID NO:1 from about nt2314 to about nt 3021, and substantial portions thereof.

The present invention further provides oligonucleotide molecules thatare useful as primers to amplify any of the aforementionedpolynucleotide molecules of the present invention or portions thereof,or that can be used to encode or act as anti-sense molecules useful inregulating ave gene expression and avermectin production.

The present invention further provides compositions and methods forcloning and expressing any of the polynucleotide molecules oroligonucleotide molecules of the present invention, including cloningvectors, expression vectors, transformed host cells comprising any ofsaid vectors, and novel strains or cell lines derived therefrom. In anon-limiting embodiment, the present invention provides a recombinantexpression vector comprising a polynucleotide molecule of the presentinvention in operative association with one or more regulatory elementsnecessary for expression of the polynucleotide molecule. In a specificthough non-limiting embodiment, the present invention provides plasmidpSE201 (ATCC 203182), which comprises the complete ORFs of both theaveR1 and aveR2 genes of S. avermitilis. Other plasmids are describedbelow.

The present invention further provides a substantially purified orisolated polypeptide encoded by a polynucleotide molecule of the presentinvention. In a specific though non-limiting embodiment, the polypeptideis an aveR1 gene product comprising the amino acid sequence of SEQ IDNO:2. In another specific though non-limiting embodiment, thepolypeptide is an aveR2 gene product comprising the amino acid sequenceof SEQ ID NO:4.

The present invention further provides substantially purified orisolated polypeptides that are homologous to either the aveR1 or aveR2gene products of the present invention. The present invention furtherprovides substantially purified or isolated peptide fragments of theaveR1 or aveR2 gene products or homologous polypeptides of the presentinvention.

The present invention further provides a method of preparing asubstantially purified or isolated aveR1 gene product, aveR2 geneproduct, homologous polypeptide, or peptide fragment of the presentinvention, comprising culturing a host cell transformed or transfectedwith a recombinant expression vector of the present invention underconditions conducive to the expression of the particular encoded geneproduct, polypeptide, or peptide fragment, and recovering the expressedgene product, polypeptide, or peptide fragment from the cell culture.

The present invention further provides compositions and methods forgenetically modifying the cells of a species or strain of Streptomyces,including genetic constructs such as, erg., gene replacement vectors. Asprovided by the present invention, the cells of a species or strain ofStreptomyces are genetically modified to produce an amount ofavermectins which is detectably different from the amount of avermectinsproduced by cells of the same species or strain that have not been somodified in a preferred embodiment, the cells of a species or strain ofStreptomyces are genetically modified to produce a detectably increasedamount of avermectins compared to the amount of avermectins produced bycells of the same species or strain that have not been so modified. In afurther preferred embodiment, the species of Streptomyces is S.avermitilis. According to the present invention, such geneticmodification preferably comprises mutating either an aveR1 homolog gene,or an aveR2 homolog gene, or both the aveR1 and aveR2 homolog genes,where such mutation results in a detectable increase in the amount ofavermectins produced by cells of a strain of Streptomyces carrying themutation compared to cells of the same strain that do not carry the genemutation. Mutation of either the aveR1 homolog gene or the aveR2 homologgene, or both aveR1 and aveR2 homolog genes, can be carried out usingstandard mutagenic techniques, including exposure to a chemical mutagenor radiation, or by using a genetic construct provided by the presentinvention, such as, e.g., a gene replacement vector, to mutate the aveR1homolog gene or aveR2 homolog gene, or both aveR1 and aveR2 homologgenes, by, e.g., adding, deleting or substituting nucleotides, or byintroducing a frame-shift, or by inserting a different or heterologousnucleotide sequence into the aveR1 homolog gene or aveR2 homolog gene,or by deleting a portion or all of either the aveR1 homolog gene or theaveR2 homolog gene, or both the aveR1 and aveR2 homolog genes, or byreplacing a portion or all of either the aveR1 homolog gene or the aveR2homolog gene, or both the aveR1 and aveR2 homolog genes, with adifferent or heterologous nucleotide sequence, or by a combination ofsuch mutations.

The present invention further provides a method for identifying amutation of an aveR1 homolog gene or aveR2 homolog gene, or of bothaveR1 and aveR2 homolog genes, in a species or strain of Streptomyces,which mutation is capable of detectably increasing the amount ofavermectins produced by cells of the species or strain of Streptomycescarrying the gene mutation compared to cells of the same species orstrain of Streptomyces that do not carry the gene mutation, comprising:(a) measuring the amount of avermectins produced by cells of theparticular species or strain of Streptomyces; (b) introducing a mutationinto the aveR1 homolog gene or aveR2 homolog gene, or into both theaveR1 and aveR2 homolog genes, of cells of the species or strain; and(c) comparing the amount of avermectins produced by the cells carryingthe gene mutation as produced in step (b) to the amount of avermectinsproduced by the cells of step (a) that do not carry the gene mutation;such that if the amount of avermectins produced by the cells carryingthe gene mutation as produced in step (b) is detectably higher than theamount of avermectins produced by the cells of step (a) that do notcarry the gene mutation, then a mutation of the aveR1 or aveR2 homologgene, or of both the aveR1 and aveR2 homolog genes, capable ofdetectably increasing the amount of avermectins has been identified. Ina preferred embodiment, the species of Streptomyces is S. avermitilis.

The present invention further provides a method of preparing geneticallymodified cells from a particular species or strain of Streptomyces,which modified cells produce a detectably increased amount ofavermectins compared to unmodified cells of the species or strain,comprising mutating the aveR1 homolog gene or the aveR2 homolog gene, orboth the aveR1 and aveR2 homolog genes, in cells of the species orstrain of Streptomyces, and selecting those cells which produce adetectably increased amount of avermectins as a result of the mutationcompared to cells of the same species or strain of Streptomyces that donot carry the gene mutation. In a preferred embodiment, the species ofStreptomyces is S. avermitilis. In a specific though non-limitingembodiment described below in Section 6.9.1, both the aveR1 and aveR2genes of S. avermitilis were mutated by replacing a portion of the ORFof each gene with a heterologous gene, resulting in S. avermitilis cellsthat produce a detectably increased amount of avermectins compared tocells of the same strain of S. avermitilis in which the aveR1 and aveR2genes have not been so mutated. In another specific though non-limitingembodiment described below in Section 6.9.2, the aveR2 gene of S.avermitilis was mutated by inserting a heterologous gene into the aveR2ORF, resulting in S. avermitilis cells that produce a detectablyincreased amount of avermectins compared to cells of the same strain ofS. avermitilis in which the aveR2 gene has not been so mutated.

The present invention further provides novel strains of Streptomyces,the cells of which produce a detectably increased amount of avermectinsas a result of one or more mutations to the aveR1 homolog gene or aveR2homolog gene, or to both the aveR1 and aveR2 homolog genes, compared tocells of the same strain of Streptomyces that do not carry the genemutation. In a preferred embodiment, the strain of Streptomyces is fromthe species S. avermitilis. The novel strains of the present inventionare useful in the large-scale production of avermectins, such as thecommercially desirable doramectin.

The present invention further provides a process for increasing theamount of avermectins produced by cultures of Streptomyces, comprisingculturing cells of a particular species or strain of Streptomyces, whichcells comprise a mutation in the aveR1 homolog gene or aveR2 homologgene, or in both the aveR1 and aveR2 homolog genes, and which genemutation serves to detectably increase the amount of avermectinsproduced by cells of the species or strain of Streptomyces carrying thegene mutation compared to cells of the same species or strain that donot carry the gene mutation, in culture media under conditions whichpermit or induce the production of avermectins therefrom, and recoveringthe avermectins from the culture. In a preferred embodiment, the speciesof Streptomyces is S. avermitilis. This process is useful to increasethe production efficiency of avermectins.

The present invention further provides antibodies directed against anaveR1 gene product, aveR2 gene product, homologous polypeptide, orpeptide fragment of the present invention.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A. Comparison of deduced amino acid sequences encoded by the S.coelicolor histidine kinase absA1 locus (SEQ ID NO:5) and the S.avermitilis aveR1 gene (SEQ ID NO:2) indicates about 32% sequenceidentity.

FIG. 1B. Comparison of deduced amino acid sequences encoded by the S.coelicolor response regulator absA2 locus (SEQ ID NO:6) and the S.avermitilis aveR2 gene (SEQ ID NO:4) indicates about 45% sequenceidentity. Highly conserved amino acids are in bold-face type.

FIG. 2A. Plasmid vector pSE201 (ATCC 203182) containing the aveR1 andaveR2 ORFs.

FIG. 2B. Plasmid vector pSE210 containing the aveR1 and aveR2 ORFs.

FIG. 3A. Gene replacement vector pSE214 containing the ermE gene, whichhas replaced a portion of the aveR1 and aveR2 ORFs.

FIG. 3B. Gene replacement vector pSE216 containing the ermE geneinserted into the aveR2 ORF.

FIG. 4. BamHI restriction map of the avermectin polyketide synthase genecluster from S. avermitilis with five overlapping cosmid clonesidentified (ie., pSE65, pSE66, pSE67, pSE68, pSE69).

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the identification and characterizationof polynucleotide molecules having nucleotide sequences that encode theaveR1 and aveR2 gene products from S. avermitilis, and the discoverythat mutation of these genes can modulate the amount of avermectinsproduced. By way of example, the invention is described in the sectionsbelow for a polynucleotide molecule comprising the nucleotide sequenceof the aveR1 ORF as shown in SEQ ID NO:1 from about nt 1112 to about nt2317, or as present in plasmid pSE201 (ATCC 203182), and for apolynucleotide molecule comprising the nucleotide sequence of the aveR2ORF as shown in SEQ ID NO:3 (note: SEQ ID NO:3 is identical to SEQ IDNO:1) from about nt 2314 to about nt 3021, or as present in plasmidpSE201 (ATCC 203182), and for polynucleotides molecules comprisingmutated nucleotide sequences derived therefrom. References herein to thenucleotide sequences shown in SEQ ID NOS:1 and 3, and to substantialportions thereof, are intended to also refer to the correspondingnucleotide sequences and substantial portions thereof, respectively, aspresent in plasmid pSE201 (ATCC 203182), unless otherwise indicated. Inaddition, references herein to the amino acid sequences shown in SEQ IDNOS:2 and 4, and to peptide fragments thereof, are intended to alsorefer to the corresponding amino acid sequences and peptide fragmentsthereof, respectively, encoded by the corresponding AveR1- andAveR2-encoding nucleotide sequences present in plasmid pSE201 (ATCC203182), unless otherwise indicated.

5.1. Polynucleotide Molecules

As used herein, the terms “polynucleotide molecule,” “polynucleotidesequence,” “coding sequence,” open-reading frame (ORF)Y, and the like,are intended to refer to both DNA and RNA molecules, which can either besingle-stranded or double-stranded. A coding sequence or ORF can includebut is not limited to prokaryotic sequences, cDNA sequences, genomic DNAsequences, and chemically synthesized DNA and RNA sequences.

Production and manipulation of the polynucleotide molecules andoligonucleotide molecules disclosed herein are within the skill in theart and can be carried out according to recombinant techniquesdescribed, among other places, in Maniatis et al., 1989, MolecularCloning, A Laboratory Manual,Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Ausubel et at, 1989, Current Protocols in MolecularBiology, Greene Publishing Associates & Wiley Interscience, NY: Sambrooket al., 1989, Molecular Cloning: A Laboratory Manual, 2d ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Innis et at(eds), 1995, PCR Strategies, Academic Press, Inc., San Diego; Erlich(ed), 1992, PCR Technology, Oxford University Press, New York; andHopwood et al., 1985, Genetic Manipulation of Streptomyces, A LaboratoryManual, John Innes Foundation, Norwich, U.K., all of which areincorporated herein by reference.

5.1.1. aveR1-Related Polynucleotide Molecules

The present invention provides an isolated polynucleotide moleculecomprising a nucleotide sequence encoding an aveR1 gene product from S.avermitilis. In a preferred embodiment, the aveR1 gene product comprisesthe amino acid sequence of SEQ ID NO:2. In a non-limiting embodiment,the isolated polynucleotide molecule of the present invention comprisesthe nucleotide sequence of the aveR1 ORF of S. avermitilis as shown inSEQ ID NO:1 from about nt 1112 to about nt 2317. In a furthernon-limiting embodiment, the isolated polynucleotide molecule of thepresent invention comprises the nucleotide sequence of SEQ ID NO:1.

The present invention further provides an isolated polynucleotidemolecule that is homologous to a polynucleotide molecule comprising thenucleotide sequence of the aveR1 ORF of S. avermitilis as shown in SEQID NO:1 from about nt 1112 to about nt 2317. The term “homologous” whenused in this respect means a polynucleotide molecule comprising anucleotide sequence: (a) that encodes the same polypeptide as SEQ IDNO:1 from about nit 1112 to about nt 2317, but that includes one or moresilent changes to the nucleotide sequence according to the degeneracy ofthe genetic code; or (b) that hybridizes to the complement of apolynucleotide molecule comprising a nucleotide sequence that encodes apolypeptide comprising the amino acid sequence of SEQ ID NO:2 undermoderately stringent conditions, i.e., hybridization to filter-bound DNAin 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C.,and washing in 0.2×SSC/0.1% SDS at 42° C. (see Ausubel et al., (eds.),1989, Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3),and is useful in practicing the invention. In a preferred embodiment,the homologous polynucleotide molecule hybridizes to the complement of apolynucleotide molecule comprising a nucleotide sequence that encodes apolypeptide comprising the amino acid sequence of SEQ ID NO:2 underhighly stringent conditions, i.e., hybridization to filter-bound DNA in0.5 M NaHPO₄, 7% SDS, 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1%SDS at 68° C. (Ausubel et al., 1989, above), and is useful in practicingthe invention. In a further preferred embodiment, the homologouspolynucleotide molecule hybridizes under highly stringent conditions tothe complement of a polynucleotide molecule comprising the nucleotidesequence of SEQ ID NO:1 from about nt 1112 to about nt 2317, and isuseful in practicing the invention. In a further preferred embodiment,the homologous polynucleotide molecule has a nucleotide sequence havingat least about 70%, more preferably at least about 80%, and mostpreferably at least about 90% sequence identity to the nucleotidesequence of SEQ ID NO: 1 from about nt 1112 to about nt 2317, asdetermined by any standard nucleotide sequence identity algorithm, suchas B ASTN (GENBANK, NCBI), and hybridizes under highly stringentconditions to the complement of such a polynucleotide molecule, and isuseful in practicing the invention.

As used herein, an aveR1-related polynucleotide molecule is “useful inpracticing the invention” where the polynucleotide molecule can be usedto introduce mutations into the aveR1 ORF of S. avermitilis bysite-directed mutagenesis, such as by homologous recombination, or toamplify a polynucleotide molecule comprising the nucleotide sequence ofthe aveRt ORF of S. avermitilis using standard amplification techniques.Such homologous polynucleotide molecules can include naturally occurringaveR1 homolog genes present in other species of Streptomyces or in otherstrains of S. avermitilis, as well as mutated aveR1 alleles, whethernaturally occurring, chemically synthesized, or genetically engineered.

The present invention further provides an isolated polynucleotidemolecule comprising a nucleotide sequence that encodes a polypeptidehaving an amino acid sequence that is homologous to the amino acidsequence of SEQ ID NO:2. As used herein to refer to polypeptides havingamino acid sequences that are homologous to the amino acid sequence ofan aveR1 gene product from S. avermitilis, the term “homologous” means apolypeptide comprising the amino acid sequence of SEQ ID NO:2, but inwhich one or more amino acid residues thereof has been conservativelysubstituted with a different amino acid residue, where the resultingpolypeptide is useful in practicing the invention. Conservative aminoacid substitutions are well-known in the art. Rules for making suchsubstitutions include those described by Dayhof, M.D., 1978, Nat Biomed.Res. Found., Washington, D.C., Vol. 5, Sup. 3, among others. Morespecifically, conservative amino acid substitutions are those thatgenerally take place within a family of amino acids that are related inthe acidity or polarity of their side chains. Genetically encoded aminoacids are generally divided into four groups: (1) acidic=aspartate,glutamate; (2) basic=lysine, arginine, histidine; (3) non-polar=alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine,tryptophan;

and (4) uncharged polar=glycine, asparagine, glutamine, cysteine,serine, threonine, tyrosine. Phenylalanine, tryptophan and tyrosine arealso jointly classified as aromatic amino acids. One or morereplacements within any particular group, e.g., of a leucine with anisoleucine or valine, or of an aspartate with a glutamate, or of athreonine with a serine, or of any other amino acid residue with astructurally related amino acid residue, e.g., an amino acid residuewith similar acidity or polarity, or with similarity in some combinationthereof, will generally have an insignificant effect on the function ofthe polypeptide. In a preferred embodiment, the homologous polypeptidehas at least about 70%, more preferably at least about 80%, and mostpreferably at least about 90% amino acid sequence identity to the aminoacid sequence of SEQ ID NO:2, as determined by any standard amino acidsequence identity algorithm, such as BLASTN (GENBANK, NCBI).

As used herein, an aveRt-related polypeptide is “useful in practicingthe invention” where the polypeptide can be used to raise antibodiesagainst an aveR1 gene product from S. avermitilis, or to screen forcompounds that modulate AveR1 activity or avermectin production inStreptomyces.

The present invention further provides an isolated polynucleotidemolecule consisting of a nucleotide sequence that is a substantialportion of any of the aforementioned aveR1-related polynucleotidemolecules of the present invention. As used herein, a “substantialportion” of an aveR1-related polynucleotide molecule means apolynucleotide molecule consisting of less than the complete codingsequence of a S. avermitilis aveR1 gene product or aveR1-relatedhomologous polypeptide of the present invention, but comprising at leastabout 20%, and more preferably at least about 30%, of said nucleotidesequence, and that is useful in practicing the invention, as usefulnessis defined above for aveR1-related polynucleotide molecules.

In a non-limiting embodiment, the substantial portion of theaveR1-related polynucleotide molecule consists of a nucleotide sequencethat encodes a peptide fragment of a S. avermitilis aveR1 gene productor aveR1-related homologous polypeptide of the present invention. A“peptide fragment” of an aveR1-related polypeptide refers to apolypeptide consisting of a sub-sequence of the amino acid sequence of afull-length aveR1 gene product or homologous polypeptide, whichsub-sequence is shorter in length than the full-length aveR1 geneproduct or homologous polypeptide, and which sub-sequence is useful inpracticing the invention, as usefulness is defined above foraveR1-related polypeptides. In a preferred embodiment, the presentinvention provides a polynucleotide molecule consisting of a nucleotidesequence encoding a peptide fragment consisting of a sub-sequence of theamino acid sequence of SEQ ID NO:2. Peptide fragments of the inventionare preferably at least about 15 amino acid residues in length.

The aveR1-related polynucleotide molecules disclosed herein can be usedto express the aveR1 gene product, to prepare novel strains ofStreptomyces in which the aveR1 gene has been mutated, and to identifyaveR1 homolog genes in other bacterial species or strains using knowntechniques. Thus, the present invention further provides an isolatedpolynucleotide molecule comprising a nucleotide sequence encoding anaveR1 homolog gene product. As used herein, an “aveR1 homolog geneproduct” is defined as a gene product encoded by an aveR1 homolog genewhich, in turn, is defined in relation to the aveR1 gene of S.avermitilis as a gene from a different species of Streptomyces or theclosely related Saccharopolyspora genus and which is recognized by thoseof skill in the art as a homolog of the aveR1 gene of S. avermitilisbased on a degree of nucleotide sequence identity greater than about80%, and which also contains the conserved active site residuestypically found in histidine kinase components of two-componentsignaling systems, and which is also closely linked to the regulatedantibiotic biosynthetic genes. For example, aveR1 homology comparisonswith eubacterial two-component systems from the Nar/Deg subgroups show100% conservation of the histidine residue (H) which is the site ofauto-phosphorylation, and the asparagine residue (N) which is requiredfor autokinase activity. As used herein, the term “aveR1 homolog gene”includes the S. avermitilis aveR1 gene itself.

Methods for identifying polynucleotide clones containing aveR1 homologgenes are known in the art. For example, a polynucleotide moleculecomprising a portion of the S. avermitilis aveR1 ORF can be detectablylabeled and used to screen a genomic library constructed from DNAderived from the organism of interest. The stringency of thehybridization conditions can be selected based on the relationship ofthe reference organism, in this example S. avermitilis, to the organismof interest. Requirements for different stringency conditions are wellknown to those of skill in the art, and such conditions will varypredictably depending on the specific organisms from which the libraryand the labeled sequences are derived. Genomic DNA libraries can bescreened for aveR1 homolog gene coding sequences using the techniquesset forth, among other places, in Benton and Davis, 1977, Science196:180, for bacteriophage libraries, and in Grunstein and Hogness,1975, Proc. Natl. Acad. Sci. USA, 72:3961-3965, for plasmid libraries,which publications are incorporated herein by reference. Polynucleotidemolecules having nucleotide sequences known to include the aveR1 ORF, asshown in SEQ ID NO:1, or oligonucleotide molecules representing portionsthereof, can be used as probes in these screening experiments.

Alternatively, oligonucleotide probes can be synthesized that correspondto nucleotide sequences deduced from the amino acid sequence of thepurified aveR1 homolog gene product.

Clones identified as containing aveR1 homolog gene coding sequences canbe tested for appropriate biological function. For example, the clonescan be subjected to sequence analysis in order to identify a suitablereading frame, as well as initiation and termination signals. The clonedDNA sequence can then be inserted into an appropriate expression vectorwhich is then transformed into cells of a strain of S. avermitilis thathave been rendered aveR1 to test for complementation. Transformed S.avermitilis host cells can then be analyzed for avermectin productionusing methods such as HPLC analysis of fermentation products, asdescribed, e.g., in Section 6.6, below.

The present invention further provides an isolated polynucleotidemolecule comprising one or more nucleotide sequences that naturallyflank the aveR1 ORF of S. avermitilis in situ. Such flanking sequencescan be selected from the nucleotide sequence of SEQ ID NO:1 from aboutnt 1 to about nt 1111, and from about nt 2318 to about nt 5045. Thepresent invention further provides an isolated polynucleotide moleculecomprising one or more nucleotide sequences that are homologous tonucleotide sequences that naturally flank the aveR1 ORF of S.avermitilis in situ. As used herein, a nucleotide sequence is homologousto a nucleotide sequence which naturally flanks the aveR1 ORF of S.avermitilis in situ where the homologous nucleotide sequence hybridizesto the complement of the nucleotide sequence which naturally flanks theaveR1 ORF of S. avermitilis in situ under moderately stringentconditions, i.e., hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7%SDS, 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1% SDS at 42° C. (seeAusubel et al., 1989, above), and is useful in practicing the invention,as usefulness is defined above for aveR1-related polynucleotidemolecules. Each flanking sequence, or homolog thereof, in the isolatedpolynucleotide molecule of the present invention is preferably at leastabout 200 nt in length. In a non-limiting embodiment, the presentinvention provides an isolated polynucleotide molecule comprising one ormore of the aforementioned nucleotide sequences that naturally flank theaveR1 ORF of S. avermitilis in situ, or that are homologous to suchnucleotide sequences, and further comprising one of the aforementionedaveR1-related nucleotide sequences of the present invention such as,e.g., the nucleotide sequence of the aveR1 ORF of S. avermitilis asshown in SEQ ID NO:1 from about nt 1112 to about nt 2317 or asubstantial portion thereof.

5.1.2. aveR2-Related Polynucleotide Molecules

The present invention further provides an isolated polynucleotidemolecule comprising a nucleotide sequence encoding an aveR2 gene productfrom S. avermitilis. In a preferred embodiment, the aveR2 gene productcomprises the amino acid sequence of SEQ ID NO:4. In a non-limitingembodiment, the isolated polynucleotide molecule of the presentinvention comprises the nucleotide sequence of the aveR2 ORF of S.avermitilis as shown in SEQ ID NO:3 from about nt 2314 to about nt 3021.In a further non-limiting embodiment, the isolated polynucleotidemolecule of the present invention comprises the nucleotide sequence ofSEQ ID NO:3.

The present invention further provides an isolated polynucleotidemolecule that is homologous to a polynucleotide molecule comprising thenucleotide sequence of the aveR2 ORF of S. avermitilis as shown in SEQID NO:3 from about nt 2314 to about nt 3021. The term “homologous” whenused in this respect means a polynucleotide molecule comprising anucleotide sequence: (a) that encodes the same polypeptide as SEQ IDNO:3 from about nt 2314 to about nt 3021, but that includes one or moresilent changes to the nucleotide sequence according to the degeneracy ofthe genetic code; or (b) that hybridizes to the complement of apolynucleotide molecule comprising a nucleotide sequence that encodes apolypeptide comprising the amino acid sequence of SEQ ID NO:4 undermoderately stringent conditions, i.e., hybridization to filter-bound DNAin 0.5 M NaHPO₄, 7% SDS, 1 mM EDTA at 65° C., and washing in0.2×SSC/0.1% SDS at 42° C. (Ausubel et al., 1989, above), and is usefulin practicing the invention. In a preferred embodiment, the homologouspolynucleotide molecule hybridizes to the complement of a polynucleotidemolecule comprising a nucleotide sequence that encodes a polypeptidecomprising the amino acid sequence of SEQ ID NO:4 under highly stringentconditions, i.e., hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7%25 SDS, 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C.(Ausubel et al., 1989, above), and is useful in practicing theinvention. In a further preferred embodiment, the homologouspolynucleotide molecule hybridizes under highly stringent conditions tothe complement of a polynucleotide molecule comprising the nucleotidesequence of SEQ ID NO:3 from about nt 2314 to about nt 3021, and isuseful in practicing the invention. In a further preferred embodiment,the homologous polynucleotide molecule has a nucleotide sequence havingat least about 70%, more preferably at least about 80%, and mostpreferably at least about 90% sequence identity to the nucleotidesequence of SEQ ID NO:3 from about nt 2314 to about nt 3021, asdetermined by any standard nucleotide sequence identity algorithm, suchas BLASTN (GENBANK, NCBI), and hybridizes under highly stringentconditions to the complement of such a polynucleotide molecule, and isuseful in practicing the invention.

As used herein, an aveR2-related polynucleotide molecule is “useful inpracticing the invention” where the polynucleotide molecule can be usedto introduce mutations into the aveR2 ORF by site-directed mutagenesis,such as by homologous recombination, or to amplify a polynucleotidemolecule comprising the nucleotide sequence of the aveR2 ORF of S.avermitilis using standard amplification techniques. Such homologouspolynucleotide molecules can include naturally occurring aveR2 genespresent in other species of Streptomyces or in other strains of S.avermitilis, as well as mutated aveR2 alleles, whether naturallyoccurring, chemically synthesized, or genetically engineered.

The present invention further provides an isolated polynucleotidemolecule comprising a nucleotide sequence that encodes a polypeptidehaving an amino acid sequence that is homologous to the amino acidsequence of SEQ ID NO:4. As used herein to refer to polypeptides havingamino acid sequences that are homologous to the amino acid sequence ofan aveR2 gene product from S. avermitilis, the term “homologous” means apolypeptide comprising the amino acid sequence of SEQ ID NO:4, but inwhich one or more amino acid residues thereof has been conservativelysubstituted with a different amino acid residue, as conservative aminoacid substitutions are defined above, where the resulting polypeptide isuseful in practicing the invention. In a preferred embodiment, thehomologous polypeptide has at least about 70%, more preferably at leastabout 80%, and most preferably at least about 90% amino acid sequenceidentity to the amino acid sequence of SEQ ID NO:4, as determined by anystandard amino acid sequence identity algorithm, such as BLASTP(GENBANK, NCBI).

As used herein, an aveR2-related polypeptide is “useful in practicingthe invention” where the polypeptide can be used to raise antibodiesagainst an aveR2 gene product from S. avermitilis, or to screen forcompounds that modulate AveR2 activity or avermectin production inStreptomyces.

The present invention further provides an isolated polynucleotidemolecule consisting of a nucleotide sequence that is a substantialportion of any of the aforementioned aveR2related polynucleotidemolecules of the present invention. As used herein, a “substantialportion” of an aveR2-related polynucleotide molecule means apolynucleotide molecule consisting of less than the complete codingsequence of a S. avermitilis aveR2 gene product or aveR2-relatedhomologous polypeptide of the present invention, but comprising at leastabout 25%, and more preferably at least about 30%, of said nucleotidesequence, and that is useful in practicing the invention, as usefulnessis defined above for aveR2-related polynucleotide molecules.

In a non-limiting embodiment, the substantial portion of theaveR2-related polynucleotide molecule consists of a nucleotide sequencethat encodes a peptide fragment of a S avermitilis aveR2 gene product oraveR2-related homologous polypeptide of the present invention. A“peptide fragment” of an aveR2-related polypeptide refers to apolypeptide consisting of a sub-sequence of the amino acid sequence of afull-length aveR2 gene product or homologous polypeptide, whichsub-sequence is shorter in length than the full-length aveR2 geneproduct or homologous polypeptide, and which sub-sequence is useful inpracticing the invention, as usefulness is defined above foraveR2-related polypeptides. In a preferred embodiment, the presentinvention provides a polynucleotide molecule consisting of a nucleotidesequence encoding a peptide fragment consisting of a sub-sequence of theamino acid sequence of SEQ ID NO:4. Peptide fragments of the inventionare preferably at least about 15 amino acid residues in length.

The aveR2-related polynucleotide molecules disclosed herein can be usedto express the aveR2 gene product, to prepare novel strains ofStreptomyces in which the aveR2 gene has been mutated, and also toidentify aveR2 homolog genes in other bacterial species or strains usingknown techniques. Thus, the present invention further provides anisolated polynucleotide molecule comprising a nucleotide sequenceencoding an aveR2 homolog gene product. As used herein, an “aveR2homolog gene product” is defined as a gene product encoded by an aveR2homolog gene which, in turn, is defined in relation to the aveR2 gene asa gene from a different species of Streptomyces, or the closely relatedSaccharopolyspora genus and which is recognized by those of skill in theart as a homolog of the aveR2 gene of S. avermitilis based on a degreeof nucleotide sequence identity greater than about 80%, and which alsocontains the conserved active site residues typically found in responseregulator components of two-component signaling systems, and which isalso closely linked to the regulated antibiotic biosynthetic genes. Forexample, aveR2 homology comparisons with eubacterial two-componentsystems from the Nar/Deg subgroup show 100% conservation of twoaspartate residues (D), one of which is the site of phosphorylation, anda conserved lysine residue (K). As used herein, the term “aveR2 homologgene” includes the S. avermitilis aveR2 gene itself.

Methods for identifying polynucleotide clones containing aveR2 homologgenes are known in the art. For example, a polynucleotide moleculecomprising a portion of the S. avermitilis aveR2 ORF can be detectablylabeled and used to screen a genomic library constructed from DNAderived from the organism of interest. The stringency of thehybridization conditions can be selected based on the relationship ofthe reference organism, in this example S. avermitilis, to the organismof interest. Genomic DNA libraries can be screened for aveR2 homologgene coding sequences using the techniques cited above in Section 5.1.1.Polynucleotide molecules having nucleotide sequences known to includethe aveR2 ORF, as shown in SEQ ID NO:3, or oligonucleotide moleculesrepresenting portions; thereof, can be used as probes in these screeningexperiments. Alternatively, oligonucleotide probes can be synthesizedthat correspond to nucleotide sequences deduced from the amino acidsequence of the purified aveR2 gene product.

Clones identified as containing aveR2 homolog gene coding sequences canbe tested for appropriate biological function. For example, the clonescan be subjected to sequence analysis in order to identify a suitablereading frame, as well as initiation and termination signals. The clonedDNA sequence can then be inserted into an appropriate expression vectorwhich is then transformed into cells of a strain of S. avermitilis thathave been rendered aveR2 to test for complementation. Transformed S.avermitilis host cells can then be analyzed for avermectin productionusing methods such as HPLC analysis of fermentation products, asdescribed, e.g., in Section 6.6, below.

The present invention further provides an isolated polynucleotidemolecule comprising one or more nucleotide sequences that naturallyflank the aveR2 ORF of S. avermitilis in situ. Such flanking sequencescan be selected from the nucleotide sequence of SEQ ID NO:3 from aboutnt 1 to about nt 2313, and from about nt 3022 to about nt 5045. Thepresent invention further provides an isolated polynucleotide moleculecomprising one or more nucleotide sequences that are homologous tonucleotide sequences that naturally flank the aveR2 ORF of S.avermitilis in situ. As used herein, a nucleotide sequence is homologousto a nucleotide sequence which naturally flanks the aveR2 ORF of S.avermitilis in situ where the homologous nucleotide sequence hybridizesto the complement of the nucleotide sequence which naturally flanks theaveR2 ORF of S. avermitilis in situ under moderately stringentconditions, i.e., hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7%SDS, 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1% SDS at 42° C. (seeAusubel et al., 1989, above), and is useful in practicing the invention,as usefulness is defined above for aveR2-related polynucleotidemolecules. Each flanking sequence, or homolog thereof, in the isolatedpolynucleotide molecule of the present invention is preferably at leastabout 200 nt in length. In a non-limiting embodiment, the presentinvention provides an isolated polynucleotide molecule comprising one ormore of the aforementioned nucleotide sequences that naturally flank theaveR2 ORF of S. avermitilis in situ, or that are homologous to suchnucleotide sequences, and further comprising one of the aforementionedaveR2-related nucleotide sequences of the present invention such as,e.g., the nucleotide sequence of the aveR2 ORF as shown in SEQ ID NO:3from about nt 2314 to about nt 3021 or a substantial portion thereof.

5.1.3. aveR1/aveR2-Related Polynucleotide Molecules

The present invention further provides an isolated polynucleotidemolecule comprising a nucleotide sequence encoding both the aveR1 andaveR2 gene products from S. avermitilis (referred to below as“aveR1/aveR2”). In a preferred embodiment, the aveR1 and aveR2 geneproducts comprise the amino acid sequences of SEQ ID NO:2 and SEQ IDNO:4, respectively. In a non-limiting embodiment, the isolatedaveR1/aveR2 polynucleotide molecule comprises the nucleotide sequence ofthe aveR1 ORF of S. avermitilis as shown in SEQ ID NO:1 from about nt1112 to about nt 2317, and the aveR2 ORF of S. avermitilis as shown inSEQ ID NO:1 from about nt 2314 to about nt 3021. In a furthernon-limiting embodiment, the isolated aveR1/aveR2 polynucleotidemolecule comprises the nucleotide sequence of SEQ ID NO:1 from about nt1112 to about nt 3021. In a further non-limiting embodiment, theisolated polynucleotide molecule comprises the nucleotide sequence ofSEQ ID NO:1. The nucleotide sequence of SEQ ID NO:1 shows a partialoverlap between the ORFs of aveR1 and aveR2; however, the presentinvention also includes aveR1aveR2 polynucleotide molecules in which theORFs for aveR1 and aveR2 do not overlap.

The present invention further provides an isolated polynucleotidemolecule that is homologous to a polynucleotide molecule comprising anucleotide sequence of the aveR1 ORF of S. avermitilis as shown in SEQID NO:1 from about nt 1112 to about nt 2317, and the aveR2 ORF of S.avermitilis as shown in SEQ ID NO:1 from about nt 2314 to about nt 3021.The term “homologous” when used in this respect means a polynucleotidemolecule comprising a nucleotide sequence: (a) that encodes the samepolypeptides as the aveR1 and aveR2 ORFs as shown in SEQ ID NO:1 fromabout nt 1112 to about nt 2317, and from about nt 2314 to about nt 3021,respectively, but that includes one or more silent changes to thenucleotide sequence according to the degeneracy of the genetic code; or(b) that hybridizes to the complement of a polynucleotide moleculecomprising a nucleotide sequence that encodes a first polypeptidecomprising the amino acid sequence of SEQ ID NO:2 and a secondpolypeptide comprising the amino acid sequence of SEQ ID NO:4, undermoderately stringent conditions, i.e., hybridization to filter-bound DNAin 0.5 M NaHPO₄, 7% SDS, 1 mM EDTA at 65° C., and washing in0.2×SSC/0.1% SDS at 42° C. (Ausubel et al., 1989, above), and is usefulin practicing the invention. In a preferred embodiment, the homologouspolynucleotide molecule hybridizes to the complement of a polynucleotidemolecule comprising a nucleotide sequence that encodes a firstpolypeptide comprising the amino acid sequence of SEQ ID NO:2 and asecond polypeptide comprising the amino acid sequence of SEQ ID NO:4,under highly stringent conditions, i.e., hybridization to filter-boundDNA in 0.5 M NaHPO₄, 7% SDS, 1 mM EDTA at 65° C., and washing in0.1×SSC/0.1% SDS at 68° C. (Ausubel et al., 1989, above), and is usefulin practicing the invention. In a further preferred embodiment, thehomologous polynucleotide molecule hybridizes under highly stringentconditions to the complement of a polynucleotide molecule comprising thenucleotide sequence of the aveR1 and aveR2 ORFs of S. avermitilis asshown in SEQ ID NO:1 from about nt 1112 to about nt 2317, and from aboutnt 2314 to about nt 3021, respectively, and is useful in practicing theinvention. In a further preferred embodiment, the homologouspolynucleotide molecule has a nucleotide sequence having at least about70%, more preferably at least about 80%, and most preferably at leastabout 90% sequence identity to the nucleotide sequence of the aveR1 andaveR2 ORFs of S. avermitilis as shown in SEQ ID NO:1 from about nt 1112to about nt 2317, and from about nt 2314 to about nt 3021, respectively,as determined by any standard nucleotide sequence identity algorithm,such as BLASTN (GENBANK, NCBI), and hybridizes under highly stringentconditions to the complement of such a polynucleotide molecule, and isuseful in practicing the invention.

As used herein, an aveR1/aveR2-related polynucleotide molecule is4useful in practicing the inventions where the polynucleotide moleculecan be used to introduce mutations into either the aveR1 ORF or aveR2ORF, or into both aveR1 and aveR2 ORFs, of S. avermitilis, bysite-directed mutagenesis, such as by homologous recombination, or toamplify a polynucleotide molecule comprising the nucleotide sequence ofeither the aveR1 ORF or aveR2 ORF, or both the aveR1 and aveR2 ORFs, ofS. avermitilis using standard amplification techniques. Such homologouspolynucleotide molecules can include naturally occurring aveR1/aveR2genes present in other species of Streptomyces or in other strains of S.avermitilis, as well as mutated aveR1 or aveR2 alleles, whethernaturally occurring, chemically synthesized, or genetically engineered.

The present invention further provides an isolated polynucleotidemolecule comprising a nucleotide sequence that encodes first and secondpolypeptides having amino acid sequences that are homologous to theamino acid sequences of SEQ ID NOS:2 and 4, respectively. As used hereinto refer to polypeptides having amino acid sequences that are homologousto the amino acid sequences of the aveR1 and aveR2 gene products from S.avermitilis, the term “homologous” means polypeptides comprising theamino acid sequences of SEQ ID NOS:2 and 4, respectively, but in whichone or more amino acid residues thereof has been conservativelysubstituted with a different amino acid residue, as conservative aminoacid substitutions are defined above, where the resulting polypeptidesare useful in practicing the invention. In a preferred embodiment, thehomologous polypeptides have at least about 70%, more preferably atleast about 80%, and most preferably at least about 90% amino acidsequence identity to the amino acid sequences of SEQ ID NO:2 and SEQ IDNO:4, respectively, as determined by any standard amino acid sequenceidentity algorithm, such as BLASTP (GENBANK, NCBI).

The present invention further provides an isolated polynucleotidemolecule consisting of a nucleotide sequence that is a substantialportion of any of the aforementioned aveR1/aveR2-related polynucleotidemolecules of the present invention. As used herein, a “substantialportion” of an aveR1/aveR2-related polynucleotide molecule means apolynucleotide molecule consisting of less than the complete codingsequence required to encode both aveR1 and aveR2 gene products from S.avermitilis, or both aveR1 and aveR2related homologous polypeptides ofthe present invention, but comprising at least about 10%, and morepreferably at least about 20%, of said nucleotide sequence, and that isuseful in practicing the invention, as usefulness is defined above foraveR1/aveR2-related polynucleotide molecules. In a preferred embodiment,the substantial portion of the aveR1/aveR2-related polynucleotidemolecule consists of a nucleotide sequence that encodes either the S.avermitilis aveR1 gene product or the S. avermitilis aveR2 gene productof the present invention, or homologous polypeptides thereof. In aspecific though non-limiting embodiment, the substantial portion of theaveR1/aveR2-related polynucleotide molecule consists of the nucleotidesequence of the aveR1 ORF as shown in SEQ ID NO:1 from about nt 1112 toabout nt 2317. In another specific though non-limiting embodiment, thesubstantial portion of the aveR1/aveR2-related polynucleotide moleculeconsists of the nucleotide sequence of the aveR2 ORF as shown in SEQ IDNO:3 from about nt 2314 to about nt 3021.

The present invention further provides an isolated polynucleotidemolecule comprising one or more nucleotide sequences that naturallyflank the aveR1/aveR2 ORFs of S. avermitilis in situ. Such flankingsequences can be selected from the nucleotide sequence of SEQ ID NO:1from about nt 1 to about nt 1111, and from about nt 3022 to about nt5045. The present invention further provides an isolated polynucleotidemolecule comprising one or more nucleotide sequences that are homologousto nucleotide sequences that naturally flank the aveR1/aveR2 ORFs of S.avermitilis in situ. As used herein, a nucleotide sequence is homologousto a nucleotide sequence which naturally flanks the aveR1/aveR2 ORFs ofS. avermitilis in situ where the homologous nucleotide sequencehybridizes to the complement of the nucleotide sequence which naturallyflanks the aveR1/aveR2 ORFs of S. avermitilis in situ under moderatelystringent conditions, i.e., hybridization to filter-bound DNA in 0.5 MNaHPO₄, 7% SDS, 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1% SDS at42° C. (see Ausubel et al., 1989, above), and is useful in practicingthe invention, as usefulness is defined above for an aveR1/aveR2-relatedpolynucleotide molecule. Each flanking sequence, or homolog thereof, inthe isolated polynucleotide molecule of the present invention ispreferably at least about 200 nt in length. In a non-limitingembodiment, the present invention provides an isolated polynucleotidemolecule comprising one or more of the aforementioned nucleotidesequences that naturally flank the aveR1/aveR2 ORFs of S. avermitilis insitu, or that are homologous to such nucleotide sequences, and furthercomprising one of the aforementioned aveR1/aveR2-related nucleotidesequences of the present invention such as, e.g., at nucleotide sequenceencoding either or both of the aveR1 and aveR2 ORFs of S. avermitilis asshown in SEQ ID NO:1 from about nt 1112 to about nt 3021.

5.2. Oligonucleotide Molecules

The present invention further provides oligonucleotide molecules thathybridize to any of the aforementioned polynucleotide molecules of thepresent invention, or that hybridize to a polynucleotide molecule havinga nucleotide sequence that is the complement of any of theaforementioned polynucleotide molecules of the present invention. Sucholigonucleotide molecules are preferably at least about 10 nucleotidesin length, but can extend up to the length of any sub-sequence of any ofthe aforementioned polynucleotide molecules of the present invention,and can hybridize to one or more of the aforementioned polynucleotidemolecules under moderately or highly stringent conditions. For shorteroligonucleotide molecules, an example of highly stringent conditionsincludes washing in 6×SSC/0.5% sodium pyrophosphate at about 37° C. for˜14-base oligos, at about 48° C. for ˜17-base oligos, at about 55° C.for ˜20-base oligos, and at about 60° C. for ˜23-base oligos. For longeroligonucleotide molecules (i.e., greater than about 100 nts), examplesof moderately and highly stringent conditions are described in Section5.1 above for homologous polynucleotide molecules.

Hybridization conditions can be appropriately adjusted as known in theart, depending upon the particular oligonucleotide molecules utilized.

In a preferred embodiment, an oligonucleotide molecule of the presentinvention hybridizes under highly stringent conditions to apolynucleotide molecule consisting of the nucleotide sequence of SEQ IDNO:1, or to a polynucleotide molecule consisting of a nucleotidesequence that is the complement of the nucleotide sequence of SEQ IDNO:1.

The oligonucleotide molecules of the present invention are useful for avariety of purposes, including as primers in amplifying an aveR1 oraveR2 gene product-encoding polynucleotide molecule, or as anti-sensemolecules useful in regulating ave genes and avermectin biosynthesis inStreptomyces. Amplification can be carried out using suitably designedoligonucleotide molecules in conjunction with standard techniques, suchas the polymerase chain reaction (PCR), although other amplificationtechniques known in the art, e.g., the ligase chain reaction, can alsobe used. For example, for PCR, a mixture comprising suitably designedprimers, a template comprising the nucleotide sequence to be amplified,and appropriate PCR enzymes and buffers, is prepared and processedaccording to standard protocols to amplify a specific aveR1- oraveR2-related polynucleotide sequence of the template.

5.3. Recombinant Expression Systems 5.3.1. Expression Vectors

The present invention further provides recombinant cloning vectors andrecombinant expression vectors comprising a polynucleotide molecule ofthe present invention, which vectors are useful in cloning or expressingsaid polynucleotide molecules, including polynucleotide moleculescomprising either the aveR1 ORF or the aveR2 ORF, or both the aveR1 andaveR2 ORFs, of S. avermitilis. In a non-limiting embodiment, the presentinvention provides plasmid pSE201 (ATCC 203182), which comprises theentire aveR1 ORF and the entire aveR2 ORF of S. avermitilis.

The following description is intended to apply to all of theaforementioned polynucleotide molecules and polypeptides of the presentinvention, including polynucleotide molecules comprising either or bothof the aveR1 and aveR2 ORFs from S. avermitilis and their gene products,and all homologous polynucleotide molecules, homologous polypeptides,substantial portions of such polynucleotide molecules, and peptidefragments of such gene products and polypeptides, as defined above,unless otherwise indicated.

A variety of different vectors have been developed for specific use inStreptomyces, including phage, high copy number plasmids, low copynumber plasmids, suicide plasmids, temperature-sensitive plasmids, and Ecoli-Streptomyces shuttle vectors, among others, and any of these can beused to practice the present invention. A number of drug resistancegenes have also been cloned from Streptomyces, and several of thesegenes have been incorporated into vectors as selectable markers.Examples of current vectors for use in Streptomyces are presented, amongother places, in Hutchinson, 1980, Applied Biochem. Biotech. 16:169-190.

Recombinant vectors of the present invention, particularly expressionvectors, are preferably constructed so that the coding sequence for thepolynucleotide molecule of the present invention is in operativeassociation with one or more regulatory elements necessary fortranscription and translation of the coding sequence to produce apolypeptide. As used herein, the term “regulatory element” includes butis not limited to nucleotide sequences that encode inducible andnoninducible promoters, enhancers, operators and other elements known inthe art that serve to drive and/or regulate expression of polynucleotidecoding sequences. Also, as used herein, the coding sequence is in“operative association” with one or more regulatory elements where theregulatory elements effectively regulate and allow for the transcriptionof the coding sequence or the translation of its mRNA, or both.

Typical plasmid vectors that can be engineered to contain apolynucleotide molecule of the present invention include pCR-Blunt,pCR2.1 (Invitrogen), pGEM3Zf (Promega), and the shuttle vector pWHM3(Vara et al., 1989, J. Bact. 171:5872-5881), among many others.

The regulatory elements of these vectors can vary in their strength andspecificities. Depending on the host/vector system utilized, any of anumber of suitable transcription and translation elements can be used.Non-limiting examples of transcriptional regulatory regions or promotersfor bacteria include the β-gal promoter, the T7 promoter, the TACpromoter, λ left and right promoters, trp and lac promoters, trp-lacfusion promoters and, more specifically for Streptomyces, the promotersermE, melC, and tipA, etc.

Methods are well-known in the art for constructing recombinant vectorscontaining particular coding sequences in operative association withappropriate regulatory elements, and any of these can be used topractice the present invention. These methods include in vitrorecombinant techniques, synthetic techniques, and in vivo geneticrecombination. See, e.g., the techniques described in Maniatis et al.,1989, above: Ausubel et al., 1989, above;

Sambrook et al., 1989, above; Innis et al., 1995, above; Erlich, 1992,above; and Hopwood et al, 1985, above.

Fusion protein expression vectors can be used to express an aveR1 or anaveR2 gene product-fusion protein. The purified fusion protein can beused to raise antisera against the aveR1 or aveR2 gene product, to studythe biochemical properties of the aveR1 or aveR2 gene product, toengineer aveR1 or aveR2 fusion proteins with different biochemicalactivities, or to aid in the identification or purification of theexpressed aveR1 or aveR2 gene product in recombinant expression systems.Possible fusion protein expression vectors include but are not limitedto vectors incorporating sequences that encode β-galactosidase and trpEfusions, maltose binding protein fusions, glutathione-S-transferasefusions and polyhistidine fusions (carrier regions).

AveR1 or AveR2 fusion proteins can be engineered to comprise a regionuseful for purification. For example, AveR1- or AveR2-maltose-bindingprotein fusions can be purified using amylose resin; AveR1- orAveR2glutathione-S-transferase fusion proteins can be purified usingglutathione-agarose beads; and AveR1or AveR2-polyhistidine fusions canbe purified using divalent nickel resin. Alternatively, antibodiesagainst a carrier protein or peptide can be used for affinitychromatography purification of the fusion protein. For example, anucleotide sequence coding for the target epitope of a monoclonalantibody can be engineered into the expression vector in operativeassociation with the regulatory elements and situated so that theexpressed epitope is fused to the AveR1 or AveR2 polypeptide. Forexample, a nucleotide sequence coding for the FLAG™ epitope tag(International Biotechnologies Inc.), which is a hydrophilic markerpeptide, can be inserted by standard techniques into the expressionvector at a point corresponding to the amino or carboxyl terminus of theAveR1 or AveR2 polypeptide. The expressed AveR1 or AveR2polypeptide-FLAG™ epitope fusion product can then be detected andaffinity-purified using commercially available anti- FLAG™ antibodies.

The expression vector encoding the AveR1 or AveR2 fusion protein canalso be engineered to contain polylinker sequences that encode specificprotease cleavage sites so that the expressed AveR1 or AveR2 polypeptidecan be released from the carrier region or fusion partner by treatmentwith a specific protease. For example, the fusion protein vector caninclude DNA sequences encoding thrombin or factor Xa cleavage sites,among others.

A signal sequence upstream from and in reading frame with the aveR1 oraveR2 ORF can be engineered into the expression vector by known methodsto direct the trafficking and secretion of the expressed gene product.Non-limiting examples of signal sequences include those from a-factor,immunoglobulins, outer membrane proteins, penicillinase, and T-celireceptors, among others.

To aid in the selection of host cells transformed or transfected withcloning or expression vectors of the present invention, the vector canbe engineered to further comprise a coding sequence for a reporter geneproduct or other selectable marker. Such a coding sequence is preferablyin operative association with the regulatory element coding sequences,as described above. Reporter genes which can be useful in the inventionare well-known in the art and include those encoding green fluorescentprotein, luciferase, xylE, and tyrosinase, among others. Nucleotidesequences encoding selectable markers are well-known in the art, andinclude those that encode gene products conferring resistance toantibiotics or anti-metabolites, or that supply an auxotrophicrequirement. Examples of such sequences include those that encoderesistance to erythromycin, thiostrepton or kanamycin, among manyothers.

5.3.2. Host Cells

The present invention further provides transformed host cells comprisinga polynucleotide molecule or recombinant vector of the invention, andnovel strains or cell lines derived therefrom. Host cells useful in thepractice of the invention are preferably Streptomyces cells, althoughother prokaryotic cells or eukaryotic cells can also be used. Suchtransformed host cells typically include but are not limited tomicroorganisms, such as bacteria transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA vectors, or yeasttransformed with recombinant vectors, among others.

Bacterial cells are generally preferred as host cells. It should beunderstood that the polynucleotide molecules of the present inventionare intended to function in Streptomyces cells, but can also betransformed into other bacterial or eukaryotic cells, e.g., for cloningor expression purposes. A strain of E. coli can typically be used, suchas, e.g., the DH5α strain, which is available either from the AmericanType Culture Collection (ATCC), Rockville, Md., USA (Accession No.31343) or from commercial sources (Stratagene). Preferred eukaryotichost cells include yeast cells, although mammalian cells or insect cellscan also be utilized effectively.

The recombinant expression vector of the invention is preferablytransformed or transfected into one or more host cells of asubstantially homogeneous culture of cells. The expression vector isgenerally introduced into host cells in accordance with knowntechniques, such as, e.g., by protoplast transformation, calciumphosphate precipitation, calcium chloride treatment, microinjection,electroporation, transfection by contact with a recombined virus,liposomemediated transfection, DEAE-dextran transfection, transduction,conjugation, or microprojectile bombardment. Selection of transformantscan be conducted by standard procedures, such as selecting for cellsexpressing a selectable marker, e.g., antibiotic resistance, associatedwith the recombinant vector, as described above.

Once the expression vector is introduced into the host cell, theintegration and maintenance of the aveR1-, aveR2- or aveR1/aveR2-relatedcoding sequence, either in the host cell chromosome or episomally, canbe confirmed by standard techniques, e.g., by Southern hybridizationanalysis, restriction enzyme analysis, PCR analysis, including reversetranscriptase PCR (rt-PCR), or by immunological assay to detect theexpected gene product. Host cells containing and/or expressing therecombinant aveR1-, aveR2- or aveR1/aveR2related coding sequence can beidentified by any of at least four general approaches which arewell-known in the art, including: (i) DNA-DNA, DNA-RNA, or RNA-antisenseRNA hybridization; (ii) detecting the presence of “marker” genefunctions; (iii) assessing the level of transcription as measured by theexpression of aveR1or aveR2-specific mRNA transcripts in the host cell;and (iv) detecting the presence of mature polypeptide product asmeasured, e.g., by immunoassay or by the presence of AveR1 or AveR2biological activity.

5.3.3. Expression and Characterization of a Recombinant aveR1 or aveR2Gene Product

Once the aveR1-, aveR2- or aveR1/aveR2-related coding sequence has beenstably introduced into an appropriate host cell, the transformed hostcell is clonally propagated, and the resulting cells are grown underconditions conducive to the maximum production of the aveR1- and/oraveR2-related gene products. Such conditions typically include growingcells to high density. Where the expression vector comprises aninducible promoter, appropriate induction conditions such as, e.g.,temperature shift, exhaustion of nutrients, addition of gratuitousinducers (e.g., analogs of carbohydrates, such asisopropyl-β-D-thiogalactopyranoside (IPTG)), accumulation of excessmetabolic by-products, or the like, are employed as needed to induceexpression.

Where the expressed aveR1- and/or aveR2-related gene product is retainedinside the host cells, the cells are harvested and lysed, and theproduct is isolated and purified from the lysate under extractionconditions known in the art to minimize protein degradation such as,e.g., at 4° C., or in the presence of protease inhibitors, or both.Where the expressed aveR1 and/or aveR2 gene product is secreted from thehost cells, the exhausted nutrient medium can simply be collected andthe product isolated therefrom.

The expressed aveR1- and/or aveR2-related gene product can be isolatedor substantially purified from cell lysates or culture medium, asappropriate, using standard methods, including but not limited to anycombination of the following methods: ammonium sulfate precipitation,size fractionation, ion exchange chromatography, HPLC, densitycentrifugation, and affinity chromatography. Where the expressed aveR1-and/or aveR2-related gene products exhibit biological activity,increasing purity of the preparation can be monitored at each step ofthe purification procedure by use of an appropriate assay. Whether ornot the expressed aveR1- and/or aveR2-related gene products exhibitbiological activity, each can be detected as based, e.g., on size, orreactivity with an antibody otherwise specific for AveR1 or AveR2, or bythe presence of a fusion tag.

The present invention thus provides a substantially purified or isolatedpolypeptide encoded by a polynucleotide molecule of the presentinvention. In a specific though non-limiting embodiment, the polypeptideis an aveR1 gene product from S. avermitilis comprising the amino acidsequence of SEQ ID NO:2. In another specific though non-limitingembodiment, the polypeptide is an aveR2 gene product from S. avermitiliscomprising the amino acid sequence of SEQ ID NO:4. The present inventionfurther provides substantially purified or isolated polypeptides thatare homologous to either the aveR1 or aveR2 gene products of the presentinvention. The present invention further provides peptide fragments ofthe aveR1 or aveR2 gene products or homologous polypeptides of thepresent invention. The substantially purified or isolated polypeptidesof the present invention are useful for a variety of purposes, such as,e.g., screening for compounds that alter aveR1 or aveR2 gene productfunction, thereby modulating avermectin biosynthesis, and for raisingantibodies directed against aveR1 or aveR2 gene products.

As used herein, a polypeptide is “substantially purified” where thepolypeptide constitutes the majority by weight of the material in aparticular preparation. Also, as used herein, a polypeptide is“isolated” where the polypeptide constitutes at least about 90 wt % ofthe material in a particular preparation.

The present invention further provides a method of preparing asubstantially purified or isolated aveR1 gene product, aveR2 geneproduct, homologous polypeptide, or peptide fragment of the presentinvention, comprising culturing a host cell transformed or transfectedwith a recombinant expression vector, said recombinant expression vectorcomprising a polynucleotide molecule comprising a nucleotide sequenceencoding the aveR1 gene product, aveR2 gene product, homologouspolypeptide, or peptide fragment, respectively, wherein the nucleotidesequence is in operative association with one or more regulatoryelements, under conditions conducive to the expression of the particulargene product, polypeptide, or peptide fragment, and recovering theexpressed gene product, polypeptide, or peptide fragment, from the cellculture in a substantially purified or isolated form.

Once an aveR1 or aveR2 gene product of sufficient purity has beenobtained, it can be characterized by standard methods, including bySDS-PAGE, size exclusion chromatography, amino acid sequence analysis,biological activity in producing appropriate products in the avermectinbiosynthetic pathway, etc. For example, the amino acid sequence of theaveR1 or aveR2 gene product can be determined using standard peptidesequencing techniques. The aveR1 or aveR2 gene product can be furthercharacterized using hydrophilicity analysis (see, e.g., Hopp and Woods,1981, Proc. Natl. Acad. Sci. USA 78:3824), or analogous softwarealgorithms, to identify hydrophobic and hydrophilic regions of the aveR1or aveR2 gene product. Structural analysis can be carried out toidentify regions of the aveR1 or aveR2 gene product that assume specificsecondary structures. Biophysical methods such as X-ray crystallography(Engstrom, 1974, Biochem. Exp. Biol. 11:7-13), computer modeling(Fletterick and Zoller (eds), 1986, in: Current Communications inMolecular Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.), and nuclear magnetic resonance (NMR) can be used to map and studysites of interaction between the aveR1 or aveR2 gene products and theirsubstrates. Information obtained from these studies can be used toselect new sites for mutation in the aveR1 or aveR2 ORFs to help developnew strains of Streptomyces having more desirable avermectin productioncharacteristics.

5.4. Construction of aveR1 and aveR2 Mutants

The present invention further provides compositions and methods forgenetically modifying the cells of a species or strain of Streptomyces,including genetic constructs such as gene replacement vectors. In apreferred embodiment, the cells of a species or strain of Streptomycesare genetically modified to produce an amount of avermectins which isdetectably different from the amount of avermectins produced by cells ofthe same species or strain that have not been so modified. In a morepreferred embodiment, the cells of a strain of S. avermitilis aregenetically modified to produce a detectably increased amount ofavermectins compared to the amount of avermectins produced by cells ofthe same strain that have not been so modified. According to the presentinvention, such genetic modification preferably comprises mutatingeither the aveR1 homolog gene, or the aveR2 homolog gene, or both theaveR1 and aveR2 homolog genes, where such mutation results in adetectable increase in the amount of avermectins produced by cells of astrain of Streptomyces carrying the gene mutation compared to cells ofthe same strain that do not carry the gene mutation. In a preferredembodiment, such genetic modification preferably comprises mutatingeither the aveR1 gene, or the aveR2 gene, or both the aveR1 and aveR2genes, of S. avermitilis, where such mutation results in a detectableincrease in the amount of avermectins produced by cells of a strain ofS. avermitilis carrying the gene mutation compared to cells of the samestrain that do not carry the gene mutation.

According to the present invention, mutations can be introduced intoeither the aveR1homolog gene or the aveR2 homolog gene, or into bothaveR1 and aveR2 homolog genes, using any techniques presently known orto be developed in the future. For example, random mutagenesis can becarried out using standard mutagenic techniques, including exposingStreptomyces cells to ultraviolet radiation or x-rays, or to chemicalmutagens such as N—methyl-N′-nitrosoguanidine, ethyl methane sulfonate,nitrous acid or nitrogen mustards, and then selecting for cellsexhibiting detectably increased avermectin production as the result ofone or more mutations in the aveR1 and/or aveR2 homolog genes. See,e.g., Ausubel, 1989, above, for a review of mutagenesis techniques.

Alternatively, mutations to the aveR1 or aveR2 homolog genes, or to boththe aveR1and aveR2 homolog genes, can be carried out in a site-directedmanner using any of a variety of known recombinant methods, includingerror-prone PCR, or cassette mutagenesis. For example, site-directedmutagenesis that takes advantage of homologous recombination can beutilized to specifically alter the ORF of either the aveR1 or aveR2homolog gene or flanking sequence thereof, or the ORF of both the aveR1and aveR2 homolog genes or flanking sequences thereof, so as tospecifically introduce one or more mutations into these genes. Inaddition, the methods described in U.S. Pat. No. 5,605,793, comprisingrandom fragmentation, repeated cycles of mutagenesis, and nucleotideshuffling, can be used to generate large libraries of polynucleotidemolecules having nucleotide sequences encoding aveR1 and/or aveR2homolog gene mutations.

Mutations to the aveR1 or aveR2 homolog genes, or to both the aveR1 andaveR2 homolog genes, that are useful in practicing the invention includeaddition, deletion or substitution, or some combination thereof, of oneor more nucleotides in either the aveR1 gene or aveR2 homolog genes, orin both the aveR1 and aveR2 homolog genes, or in flanking regulatoryregions, and which produce the desired result, i.e., a detectableincrease in the amount of avermectins produced by cells of a strain ofStreptomyces carrying the gene mutation compared to cells of the samespecies or strain of Streptomyces which do not carry the gene mutation.Such mutations can serve to introduce one or more novel restrictionsites, termination codons, or frame shifts, into either or both of theORF sequences or into the flanking regulatory regions involved in genetranscription. Other useful mutations include those which insert adifferent or heterologous nucleotide sequence into either or both of theaveR1 and aveR2 homolog genes; or which delete all or a portion ofeither or both of the aveR1 and aveR2 homolog genes; or which replaceall or a portion of either or both of the aveR1 and aveR2 homolog geneswith a different or heterologous nucleotide sequence; and whichmutations produce the desired result, i.e., a detectable increase in theamount of avermectins produced by cells of a species or strain ofStreptomyces carrying the gene mutation compared to cells of the samespecies or strain of Streptomyces which do not carry the gene mutation.In a preferred embodiment, the mutation serves to inactive the aveR1 oraveR2 homolog gene, or both the aveR1 and aveR2 homolog genes. In a morepreferred embodiment, the mutation serves to inactivate either the aveR2homolog gene, or both the aveR1 and aveR2 homolog genes.

Site-directed mutations are useful, particularly where they serve toalter one or more conserved amino acid residues in either the aveR1 oraveR2 homolog gene product, or in both the aveR1 and aveR2 homolog geneproducts. For example, a comparison of the deduced amino acid sequencesof aveR1 and aveR2 gene products from S. avermitilis with analogous geneproducts from S. coelicolor, as presented in FIGS. 1A and 1B, indicatessites of significant conservation of amino acid residues between thesespecies. Site-directed mutagenesis which deletes or non-conservativelysubstitutes one or more of these conserved amino acid residues can beparticularly effective in producing novel mutant strains that exhibitdesirable alterations in avermectin production.

In a preferred embodiment, one or more mutations are introduced byhomologous recombination into either the aveR1 or aveR2 homolog gene, orinto both the aveR1 and aveR2 homolog genes, using a genetic constructprovided by the present invention, such as, e.g., a gene replacementvector. The genetic construct can comprise the entire ORF of the aveR1homolog gene or a homologous polynucleotide molecule thereof, or asubstantial portion thereof, or the entire ORF of the aveR2 homolog geneor a homologous polynucleotide molecule thereof, or a substantialportion thereof, or the entire ORFs of both the aveR1 and aveR2 homologgenes or a homologous polynucleotide molecule thereof, or a substantialportion thereof; or nucleotide sequences that naturally flank the ORF ofeither the aveR1 homolog gene or aveR2 homolog gene, or both the aveR1and aveR2 homolog genes in situ; or a combination thereof, and whichgenetic construct can be used to introduce a mutation into either theaveR1 homolog gene or the aveR2 homolog gene, or into both the aveR1 andaveR2 homolog genes, which mutation results in a detectable increase inthe amount of avermectins produced by cells of a species or strain ofStreptomyces carrying said gene mutation compared to cells of the samespecies or strain which do not carry the gene mutation.

In a specific though non-limiting embodiment, a genetic construct foruse in practicing the present invention is a plasmid which comprises apolynucleotide molecule comprising a nucleotide sequence that isotherwise the same as the nucleotide sequence of the ORF of either theaveR1 or aveR2 homolog gene, or both the aveR1 and aveR2 homolog genes,or a substantial portion thereof, from Streptomyces, but which furthercomprises one or more mutations, ie., one or more nucleotide deletions,insertions, substitutions, or a combination thereof, which plasmid canbe used to transform cells of Streptomyces, and thereby introduce themutation into the aveR1 homolog gene, aveR2 homolog gene, or into boththe aveR1 and aveR2 homolog genes, so as to disrupt or otherwise alterthe activity or biological function of either the aveR1 homolog gene, oraveR2 homolog gene, or both the aveR1 and aveR2 homolog genes,respectively, or to disrupt or otherwise alter the activity orbiological function of the aveR1 homolog gene product, or aveR2 homologgene product, or both aveR1 and aveR2 homolog gene products,respectively, such that the amount of avermectins produced by cells of aspecies or strain of Streptomyces carrying said gene mutation will bedetectably increased compared to cells of the same species or strainwhich do not carry the gene mutation. Such a plasmid preferably furthercomprises a selectable marker.

Once transformed into host cells of Streptomyces, the polynucleotidemolecule of the genetic construct is specifically targeted by homologousrecombination to the aveR1 homolog gene or aveR2 homolog gene, or toboth the aveR1 and aveR2 homolog genes, and either replaces the aveR1homolog gene or a portion thereof, or the aveR2 homolog gene or aportion thereof, or both the aveR1 and aveR2 homolog genes or a portionthereof, or inserts into the aveR1 homolog gene or aveR2 homolog gene.As a result of this recombination event, either the aveRt homolog geneor the aveR2 homolog gene, or both the aveR1 and aveR2 homolog genes, ofthe host cell, or the gene products encoded thereby, are partially orcompletely disabled. Transformed cells are selected, preferably bytaking advantage of the presence of a selectable marker in the geneticconstruct, and screened by standard techniques, such as those describedin Section 6.6 below, for those cells that produce a detectablyincreased amount of avermectins compared to cells of the same strainwhich have not be so transformed.

In a specific though non-limiting embodiment exemplified in Section6.9.1 below, a gene replacement vector was used to disrupt both theaveR1 and aveR2 genes of S. avermitilis by replacing a portion of theORF of each gene with a heterologous nucleotide sequence (ermE). Inanother specific though non-limiting embodiment exemplified in Section6.9.2 below, a gene replacement vector was used to disrupt the aveR2gene of S. avermitilis by inserting a heterologous nucleotide sequence(ermE) into the aveR2 ORF. Each of these gene replacement vectors wereseparately transformed into cells of a strain of S. avermitilis andintegrated into the chromosome by homologous recombination. Fermentationanalysis of each of these novel S. avermitilis transformants indicated asignificant increase in the amount of avermectins produced by cellscarrying these gene mutations compared to cells of the same strain thatdo not carry either of these gene mutations.

The present invention further provides a method for identifying amutation of an aveR1 homolog gene or aveR2 homolog gene, or of bothaveR1 and aveR2 homolog genes, in a species or strain of Streptomyces,which gene mutation is capable of detectably increasing the amount ofavermectins produced by cells of the species or strain of Streptomycescarrying the gene mutation compared to cells of the same species orstrain of Streptomyces that do not carry the gene mutation, comprising:(a) measuring the amount of avermectins produced by cells of aparticular species or strain of Streptomyces; (b) introducing a mutationinto the aveR1 homolog gene or aveR2 homolog gene, or into both theaveR1 and aveR2 homolog genes, of cells of the species or strain ofStreptomyces of step (a); and (c) comparing the amount of avermectinsproduced by the cells carrying the gene mutation as produced in step (b)to the amount of avermectins produced by the cells of step (a) that donot carry the gene mutation; such that if the amount of avermectinsproduced by the cells carrying the gene mutation as produced in step (b)is detectably higher than the amount of avermectins produced by thecells of step (a) that do not carry the gene mutation, then a mutationof the aveR1 or aveR2 homolog gene, or of both the aveR1 and aveR2homolog genes, capable of detectably increasing the amount ofavermectins produced has been identified. In a preferred embodiment, thespecies of Streptomyces is S. avermitilis.

The present invention further provides a method of preparing geneticallymodified cells from a species or strain of Streptomyces, which modifiedcells Produce a detectably increased amount of avermectins compared tounmodified cells of the same species or strain, comprising mutating theaveR1 homolog gene or the aveR2 homolog gene, or both the aveR1and aveR2homolog genes of Streptomyces, in cells of the species or strain ofStreptomyces, and selecting the mutated cells which produce a detectablyincreased amount of avermectins compared to cells of the same species orstrain of Streptomyces that do not carry the gene mutation. In apreferred embodiment, the species of Streptomyces is S. avermitilis.

The present invention further provides novel strains of Streptomyces,the cells of which produce a detectably increased amount of avermectinsas a result of one or more mutations to the aveR1 homolog gene or aveR2homolog gene, or to both the aveR1 and aveR2 homolog genes, compared tocells of the same species or strain of Streptomyces that do not carrythe gene mutation. In a preferred embodiment, the species ofStreptomyces is S. avermitilis. The novel strains of the presentinvention are useful in the large-scale production of avermectins, suchas the commercially desirable doramectin.

The present invention further provides a process for producing anincreased amount of avermectins produced by cultures of Streptomyces,comprising culturing cells of a species or strain of Streptomyces, whichcells comprise a mutation in the aveR1 homolog gene or aveR2 homologgene, or in both the aveR1 and aveR2 homolog genes, which gene mutationserves to detectably increase the amount of avermectins produced bycells of the species or strain of Streptomyces carrying the genemutation compared to cells of the same species or strain that do notcarry the gene mutation, in culture media under conditions which permitor induce the production of avermectins therefrom, and recovering theavermectins from the culture. In a preferred embodiment, the species ofStreptomyces is S. avermitilis. This process is useful to increase theproduction efficiency of avermectins.

5.5. Anti-Sense Oligonucleotides and Ribozymes

Also within the scope of the present invention are oligonucleotidesequences that include anti-sense oligonucleotides, phosphorothioatesand ribozymes that function to bind to, degrade and/or inhibit thetranslation of aveR1 or aveR2 homolog gene mRNA.

Anti-sense oligonucleotides, including anti-sense RNA molecules andanti-sense DNA molecules, act to directly block the translation of mRNAby binding to targeted mRNA and preventing protein translation. Forexample, antisense oligonucleotides of at least about 15 bases andcomplementary to unique regions of the DNA sequence encoding an AveR1 orAveR2 homolog polypeptide can be synthesized, e.g., by conventionalphosphodiester techniques.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. The mechanism of ribozyme action involves sequencespecific hybridization of the ribozyme molecule to complementary targetRNA, followed by endonucleolytic cleavage. Engineered hammerhead motifribozyme molecules that specifically and efficiently catalyzeendonucleolytic cleavage of aveR1 or aveR2 homolog mRNA sequences arealso within the scope of the present invention.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences, GUA, GUU, and GUC.Once identified, short RNA sequences of between about 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site can be evaluated for predicted structuralfeatures such as secondary structure that may render the oligonucleotidesequence unsuitable. The suitability of candidate targets can also beevaluated by testing their accessibility to hybridization withcomplementary oligonucleotides, using, e.g., ribonuclease protectionassays.

Both the anti-sense oligonucleotides and ribozymes of the presentinvention can be prepared by known methods. These include techniques forchemical synthesis such as, e.g., by solid phase phosphoamite chemicalsynthesis. Alternatively, anti-sense RNA molecules can be generated byin vitro or in vivo transcription of DNA sequences encoding the RNAmolecule. Such DNA sequences can be incorporated into a wide variety ofvectors which incorporate suitable RNA polymerase promoters such as theT7 or SP6 polymerase promoters.

Various modifications to the oligonucleotides of the present inventioncan be introduced as a means of increasing intracellular stability andhalf-life. Possible modifications include but are not limited to theaddition of flanking sequences of ribonucleotides ordeoxyribonucleotides to the 5′ and/or 3′ ends of the molecule, or theuse of phosphorothioate or 2′-O-methyl rather than phosphodiesteraselinkages within the oligonucleotide backbone.

5.6. Antibodies

The present invention further provides polyclonal and monoclonalantibodies that bind to an aveR1 homolog gene product, aveR2 homologgene product, or to an homologous polypeptide, or peptide fragment ofthe present invention. Such antibodies can be used as affinity reagentswith which to purify a native aveR1 or aveR2 homolog gene product, or toanalyze the activity or biological function of the aveR1 or aveR2homolog gene products.

Antibodies can be raised against any of the aveR1- or aveR2-relatedpolypeptides of the present invention. Various host animals, includingbut not limited to cows, horses, rabbits, goats, sheep, and mice, can beused according to known methods to produce anti-AveR1 oranti-AveR2-specific antibodies. Various adjuvants known in the art canbe used to enhance antibody production.

Polyclonal antibodies can be obtained from immunized animals and testedfor anti-AveR1 or anti-AveR2 specificity using standard techniques.Alternatively, monoclonal antibodies to an AveR1 or AveR2 polypeptidecan be prepared using any technique that provides for the production ofantibody molecules by continuous cell lines in culture. These includebut are not limited to the hybridoma technique originally described byKohler and Milstein (Nature, 1975, 256: 495-497); the human B-cellhybridoma technique (Kosbor, et al., 1983, Immunology Today 4:72; Cote,et al., 1983, Proc. Natl. Acad. Sci. USA 80: 2026-2030); and theEBV-hybridoma technique (Cole, et al., 1985, Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96). Alternatively,techniques described for the production of single chain antibodies (see,e.g., U.S. Pat. No. 4,946,778) can be adapted to produce AveR1- orAveR2-specific single chain antibodies. These publications areincorporated herein by reference.

Antibody fragments that contain specific binding sites for an AveR1 orAveR2 polypeptide are also encompassed within the present invention, andcan be generated by known techniques. Such fragments include but are notlimited to F(ab′)₂ fragments which can be generated by pepsin digestionof an intact antibody molecule, and Fab fragments which can be generatedby reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries can be constructed (Huse et al.,1989, Science 246: 1275-1281) to allow rapid identification of Fabfragments having the desired specificity to the AveR1 or AveR2polypeptide.

Techniques for the production of monoclonal antibodies and antibodyfragments are well-known in the art, and are additionally described,among other places, in Harlow and Lane, 1988, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory; and in J. W. Goding, 1986,Monoclonal Antibodies: Principles and Practice, Academic Press, London.All of the above-cited publications are incorporated herein byreference.

5.7. Uses of Avermectins

Avermectins are highly active antiparasitic agents having particularutility as anthelmintics, ectoparasiticides, insecticides andacaricides, and avermectins prepared using the compositions and methodsof the present invention are useful for any of these purposes.

The avermectins prepared according to the present invention are usefulto treat various diseases or conditions in humans, particularly wherethose diseases or conditions are caused by parasitic infections, asknown in the art. See, e.g., Ikeda and Omura, 1997, Chem. Rev.97(7):2591-2609.

The avermectins prepared according to the present invention areeffective in treating a variety of conditions caused by endoparasitesincluding, e.g., helminthiasis, which is most frequently caused by agroup of parasitic nematodes, and which can cause disease in humans, andsevere economic losses in swine, sheep, poultry, horses and cattle, aswell as affecting the health of domestic animals. Thus, avermectinsprepared according to the present invention are effective againstnematodes that affect humans, as well as those that affect variousspecies of animals including, e.g., Dirofilaria in dogs, and variousparasites which infect humans, including gastrointestinal parasites suchas Ancylostoma, Necator, Ascaris, Strongyloides, Trinchinella,Capillaria, Trichuris, Enterobius, and parasites which are found in theblood or other tissues or organs, such as filarial worms and the extractintestinal states of Strongyloides and Trichinella.

The avermectins prepared according to the present invention are alsouseful in treating ectoparasitic infections including, e.g., arthropodinfestations of mammals and birds, caused by ticks, mites, lice, fleas,blowflies, biting insects, or migrating dipterous larvae which canaffect cattle and horses, among others.

The avermectins prepared according to the present invention are alsouseful as insecticides against household pests such as, e.g., thecockroach, clothes moth, carpet beetle and the housefly among others, aswell as insect pests of stored grain and of agricultural plants, whichpests include spider mites, aphids, caterpillars, and orthopterans suchas locusts, among others.

Animals that can be treated with the avermectins of the presentinvention include sheep, cattle, horses, deer, goats, swine, birdsincluding poultry, and dogs and cats.

The avermectins prepared according to the present invention areadministered in a formulation appropriate to the specific intended use,the particular species of host animal being treated, and the parasite orinsect involved. For use as a parasiticide, an avermectin of the presentinvention can be administered orally in the form of a capsule, bolus,tablet or liquid drench or, alternatively, can be administered as apour-on, or by injection, or as an implant.

Such formulations are prepared in a conventional manner in accordancewith standard veterinary practice. Thus, capsules, boluses or tabletscan be prepared by mixing the active ingredient with a suitable finelydivided diluent or carrier additionally containing a disintegratingagent and/or binder such as starch, lactose, talc, magnesium stearate,etc. A drench formulation can be prepared by dispersing the activeingredient in an aqueous solution together with a dispersing or wettingagent, etc. Injectable formulations can be prepared in the form of asterile solution which can contain other substances such as, e.g.,sufficient salts and/or glucose to make the solution isotonic withblood.

Such formulations will vary with regard to the weight of active compounddepending on the patient, or on the species of host animal to betreated, the severity and type of infection, and the body weight of thehost. Generally, for oral administration a dose of active compound offrom about 0.001 to 10 mg per kg of patient or animal body weight givenas a single dose or in divided doses for a period of from 1 to 5 dayswill be satisfactory. However, there can be instances where higher orlower dosage ranges are indicated, as determined, e.g., by a physicianor veterinarian, as based on clinical symptoms.

As an alternative, an avermectin prepared according to the presentinvention can be administered in combination with animal feedstuff, andfor this purpose a concentrated feed additive or premix can be preparedfor mixing with the normal animal feed.

For use as an insecticide, and for treating agricultural pests, thecompounds of the present invention can be applied as sprays, dusts,emulsions and the like in accordance with standard agriculturalpractice.

The following examples are illustrative only, and are not intended tolimit the scope of the present invention.

6. EXAMPLE Isolation of aveR1 and aveR2 Genes

This example describes the isolation and characterization of two novelS. avermitilis genes that encode aveR1 and aveR2 gene products and thatare involved in the regulation of avermectin biosynthesis.

6.1. Growth of S. avermitilis for DNA Isolation

Single colonies of S. avermitilis ATCC 31272 (single colony isolate #2)were isolated on ½ strength YPD-6 medium containing: Difco YeastExtract—5 g; Difco Bacto-peptone—5 g; Dextrose—2.5 g; MOPS—5 g; DifcoBacto agar—15 g. Final volume was adjusted to 1 liter with dH₂O, pH wasadjusted to 7.0, and the medium was autoclaved at 121° C. for 25 min.The mycelia grown in the above medium were used to inoculate 10 ml ofTSB medium (Difco Tryptic Soy Broth in 1 liter dH₂O, autoclaved at 121°C. for 25 min) in a 25 mm×150 mm tube which was maintained with shaking(300 rpm) at 28° C. for 48-72 hrs.

6.2. Chromosomal DNA Isolation from S. avermitilis

Aliquots (0.25 ml or 0.5 ml) of mycelia grown as described above wereplaced in 1.5 ml microcentrifuge tubes and the cells were concentratedby centrifugation at 12,000×g for 60 sec. The supernatant was discardedand the cells were resuspended in 0.25 ml TSE buffer (20 ml 1.5 Msucrose, 2.5 ml 1 M Tris HCl, pH 8.0, 2.5 ml 1 M EDTA, pH 8.0, and 75 mldH₂O) containing 2 mg/ml lysozyme. The samples were incubated at 37° C.for 20 min with shaking, loaded into an AutoGen 540™ automated nucleicacid isolation instrument (Integrated Separation Systems, Natick, Mass.)and genomic DNA was isolated using Cycle 159 (equipment software)according to manufacturer's instructions.

Alternatively, 5 ml of mycelia were placed in a 17 mm×100 mm tube, thecells were concentrated by centrifugation at 3,000 rpm for 5 min, andthe supernatant was removed. Cells were resuspended in 1 ml TSE buffer,concentrated by centrifugation at 3,000 rpm for 5 min and thesupernatant was removed. Cells were resuspended in 1 ml TSE buffercontaining 2 mg/ml lysozyme and incubated at 37° C. with shaking for30-60 min. After incubation, 0.5 ml 10% SDS was added and the cellsincubated at 37° C. until lysis was complete. The lysate was incubatedat 65° C. for 10 min, cooled to rm temp, split into two 1.5 ml Eppendorftubes and extracted 1× with 0.5 ml phenol/chloroform (50% phenolpreviously equilibrated with 0.5 M Tris, pH 8.0; 50% chloroform). Theaqueous phase was removed and extracted 2-5× with chloroform:isoamylalcohol (24:1). The DNA was precipitated by adding {fraction (1/10)}volume 3M sodium acetate, pH 4.8, incubating the mixture on ice for 10min, centrifuging the mixture at 15,000 rpm at 5° C. for 10 min, andremoving the supernatant to a clean tube to which 1 volume ofisopropanol was added. The mixture was then incubated on ice for 20 min,centrifuged at 15,000 rpm for 20 min at 5° C., the supernatant wasremoved, and the DNA pellet was washed 1× with 70% ethanol. After thepellet was dry, the DNA was resuspended in TE buffer (10 mM Tris, 1 mMEDTA, pH 8.0).

6.3. Plasmid DNA Isolation from S. avermitilis

An aliquot (1.0 ml) of mycelia was placed in 1.5 ml microcentrifugetubes and the cells were concentrated by centrifugation at 12,000×g for60 sec. The supernatant was discarded, the cells were resuspended in 1.0ml 10.3% sucrose and concentrated by centrifugation at 12,000×g for 60sec, and the supernatant was discarded. The cells were then resuspendedin 0.25 ml TSE buffer containing 2 mg/ml lysozyme, incubated at 37° C.for 20 min with shaking, and loaded into the AutoGen 540™ automatednucleic acid isolation instrument. Plasmid DNA was isolated using Cycle106 (equipment software) according to manufacturer's instructions.

Alternatively, 1.5 ml of mycelia were placed in 1.5 ml microcentrifugetubes and the cells were concentrated by centrifugation at 12,000×g for60 sec. The supernatant was discarded, the cells were resuspended in 1.0ml 10.3% sucrose and concentrated by centrifugation at 12,000×g for 60sec, and the supernatant was discarded. The cells were resuspended in0.5 ml TSE buffer containing 2 mg/ml lysozyme and incubated at 37° C.for 1530 min. After incubation, 0.25 ml alkaline SDS (0.3N NaOH, 2% SDS)was added and the cells incubated at 55° C. for 15-30 min or until thesolution was clear. Sodium acetate (0.1 ml, 3M, pH 4.8) was then addedto the DNA solution, which was incubated on ice for 10 min. The DNAsamples were centrifuged at 14,000 rpm for 10 min at 5° C. Thesupernatant was removed to a clean tube, and 0.2 ml phenol:chloroform(50% phenol:50% chloroform) was added and gently mixed. The DNA solutionwas centrifuged at 14,000 rpm for 10 min at 5° C. and the upper layerwas removed to a clean Eppendorf tube. Isopropanol (0.75 ml) was added,and the solution was gently mixed and then incubated at rm temp for 20min. The DNA solution was centrifuged at 14,000 rpm for 15 min at 5° C.,the supernatant was removed, the DNA pellet was washed with 70% ethanol,dried, and the DNA was resuspended in TE buffer.

6.4. Plasmid DNA Isolation from E. coli

A single transformed E. coli colony was inoculated into 5 mlLuria-Bertani (LB) medium (Bacto-Tryptone—10g; Bacto-yeast extract—5g;and NaCl—10 g in 1 liter dH₂O, pH 7.0, autoclaved at 121° C. for 25 min)supplemented with 100 jig/ml ampicillin. The culture was incubatedovernight, and a 1 ml aliquot placed in a 1.5 ml microcentrifuge tube.The culture samples were loaded into the AutoGen 540™ automated nucleicacid isolation instrument and plasmid DNA was isolated using Cycle 3(equipment software) according to manufacturer's instructions.

6.5. Preparation and Transformation of S. avermitilis Protoplasts

Single colonies of S. avermitilis were isolated on ½ strength YPD-6. Themycelia were used to inoculate 10 ml of TSB medium in a 25 mm×150 mmtube which was then incubated with shaking (300 rpm) at 28° C. for 48hrs. One ml of mycelia was used to inoculate 50 ml YEME medium. YEMEmedium contains per liter Difco Yeast Extract—3 9; Difco Bacto-peptone—5g; Difco Malt Extract—3 g; and sucrose—300 9. After autoclaving at 121°C. for 25 min, the following were added: 2.5 M MgCl₂.6H₂O (separatelyautoclaved at 121° C. for 25 min)—2 ml; and glycine (20%)(filter-sterilized)—25 ml.

The mycelia were grown at 30° C. for 48-72 hrs and harvested bycentrifugation in a 50 ml centrifuge (Falcon) tube at 3,000 rpm for 20min. The supernatant was discarded and the mycelia were resuspended in Pbuffer which contains: sucrose—205 g; K₂SO₄—0.25 g; MgCl₂.6H₂O—2.02 g;H₂O—600 ml; K₂PO₄ (0.5%)—10 ml; Trace element solution*—20 ml;CaCl₂.2H₂O (3.68%)—100 ml; MES buffer (1.0 M, pH 6.5)—10 ml. (*Traceelement solution contains per liter: ZnCl₂—40 mg; FeCl₃.6H₂O—200 mg;CuCl₂.2H₂O—10 mg; MnCl₂.4H₂O—10 mg; Na₂B₄O₇.10H₂O—10 mg; (NH₄)₆Mo₇O₂₄.4H₂O—10 mg). The pH was adjusted to 6.5, final volume wasadjusted to 1 liter, and the medium was filtered hot through a 0.45micron filter.

The mycelia were pelleted at 3,000 rpm for 20 min, the supernatant wasdiscarded, and the mycelia was resuspended in 20 ml P buffer containing2 mg/ml lysozyme. The mycelia were incubated at 35° C. for 15 min withshaking, and checked microscopically to determine extent of protoplastformation. When protoplast formation was complete, the protoplasts werecentrifuged at 8,000 rpm for 10 min. The supernatant was removed and theprotoplasts were resuspended in 10 ml P buffer. The protoplasts werecentrifuged at 8,000 rpm for 10 min, the supernatant was removed, theprotoplasts were resuspended in 2 ml P buffer, and approximately 1×10⁹protoplasts were distributed to 2.0 ml cryogenic vials (Nalgene).

A vial containing 1×10⁹ protoplasts was centrifuged for 10 min at 8,000rpm, the supernatant was removed, and the protoplasts were resuspendedin 0.1 ml P buffer. Two to 5 μg of transforming DNA were added to theprotoplasts, immediately followed by the addition of 0.5 ml working Tbuffer. T buffer base contains: PEG 1000 (Sigma)—25 g; sucrose—2.5 g; 30and H₂O—83 ml. The pH was adjusted to 8.8 with 1 N NaOH(filter-sterilized), and the T buffer base was filter-sterilized andstored at 4° C. Working T buffer, made the same day used, contains Tbuffer base—8.3 ml; K₂PO₄ (4 mM)—1.0 ml; CaCl₂.2H₂O (5 M)—0.2 ml; andTES (1 M, pH 8)—0.5 ml. Each component of the working T buffer wasfilter-sterilized and stored at 4° C.

Within 20 sec of adding T buffer to the protoplasts, 1.0 ml P buffer wasalso added and the protoplasts were centrifuged at 8,000 rpm for 10 min.The supernatant was discarded and the protoplasts were resuspended in0.1 ml P buffer. The protoplasts were then plated on RM14 media whichcontains: sucrose—205 g; K₂SO₄—0.25 g; MgCl₂.6H₂O—10.12 g; glucose—10 g;Difco Casamino Acids—0.1 g; Difco Yeast Extract—5 g; Difco Oatmeal Agar3 g; Difco Bacto Agar—22 g; and H₂O—800 ml. The solution was autoclavedat 121° C. for 25 min. After autoclaving, sterile stocks of thefollowing were added: K₂PO₄ (0.5%)—10 ml; CaCl₂.2H₂O (5 M)—5 ml;L-proline (20%)—15 ml; MES buffer (1.0 M, pH 6.5)—10 ml; Trace elementsolution (same as above)—2 ml; cycloheximide stock (25 mg/ml)—40 ml; and1N NaOH—2 ml. Twenty-five ml of RM14 medium were aliquoted per plate,and plates were dried for 24 hrs before use.

The protoplasts were incubated in 95% humidity at 30° C. for 20-24 hrs.To select erythromycin-resistant transformants, 1 ml of overlay bufferplus 125 μg erythromycin (to give a final concentration of 5 pg/mlerythromycin) was spread evenly over the RM14 regeneration plates.Overlay buffer contains per 100 ml: sucrose—10.3 g; Trace elementsolution (same as above)—0.2 ml; and MES (1 M, pH 6.5)—1 ml. Theprotoplasts were incubated in 95% humidity at 30° C. for 7-14 days untilerythromycin-resistant (Erm′) colonies were visible.

6.6. Fermentation Analysis of S. avermitilis Strains

S. avermitilis mycelia grown on ½ strength YPD6 for 4-7 days wereinoculated into 1×6 inch tubes containing 8 ml of preform medium and two5 mm glass beads. Preform medium contains: soluble starch (either thinboiled starch or KOSO, Japan Corn Starch Co., Nagoya)—20 g/L;Pharmamedia (Trader's Protein, Memphis Tenn.)—15 g/L; Ardamine pH(Champlain, Inc. Clifton, N.J.)—5 g/L; CaCO₃—2 g/L; 2×bcfa (“bcfa”refers to branched chain fatty acids) containing a final concentrationin the medium of 50 ppm 2-(±)-methyl butyric acid, 60 ppm isobutyricacid, and 20 ppm isovaleric acid. The pH was adjusted to 7.2, and themedium was autoclaved at 121° C. for 25 min.

The tube was shaken at a 17° angle at 215 rpm at 29° C. for 3 days. A2-ml aliquot of the seed culture was used to inoculate a 300 mlErlenmeyer flask containing 25 ml of production medium which contains:starch (either thin boiled starch or KOSO)—160 g/L; Nutrisoy (ArcherDaniels Midland, Decatur, Ill.)—10 g/L; Ardamine pH—10 g/L; K₂HPO₄—2g/L; MgSO₄.4H₂O—2 g/L; FeSO₄.7H₂O—0.02 g/L; MnCl₂—0.002 g/L;ZnSO₄.7H₂O—0.002 g/L; CaCO₃—14 g/L; and 2×bcfa (as above). The pH wasadjusted to 6.9 and the medium was autoclaved at 121° C. for 25 min.

After inoculation, the flask was incubated at 29° C. for 12 days withshaking at 200 rpm. After incubation, a 2 ml sample was withdrawn fromthe flask, diluted with 8 ml of methanol, mixed, and the mixture wascentrifuged at 1250×g for 10 min to pellet debris. The supernatant wasthen assayed by high performance liquid chromatography (HPLC) using aBeckman Ultrasphere ODS column (25 cm×4.6 mm ID) with a flow rate of0.75 ml/min and detection by absorbance measurements at 240 nm. Themobile phase was 86/8.9/5.1 methanol/water/acetonitrile.

6.7. Identification and Isolation of S. avermitilis PKS Genes

A cosmid library of S. avermitilis (ATCC 31272) chromosomal DNA wasprepared and hybridized with a ketosynthase (KS) probe made from afragment of the Saccharopolyspora erythraea polyketide synthase (PKS)gene. A detailed description of the preparation of cosmid libraries canbe found in Sambrook et a., 1989, above. A detailed description of thepreparation of Streptomyces chromosomal DNA libraries is presented inHopwood et al., 1985, above. Cosmid clones containingketosynthase-hybridizing regions were identified by hybridization to a2.7 Kb Ndel/Eco47III fragment from pEX26 (kindly supplied by Dr. P.Leadley, Cambridge, UK). Approximately 5 jig of pEX26 were digestedusing the restriction endonucleases Ndel and Eco47III. The reactionmixture was loaded on a 0.8% SeaPlaque™ GTG agarose gel (FMCBioProducts, Rockland, Me.). The 2.7 Kb Ndel/Eco47III fragment wasexcised from the gel after electrophoresis and the DNA was recoveredfrom the gel using GELase™ (Epicentre Technologies) using the FastProtocol. The 2.7 Kb Ndel/Eco47III fragment was labeled with [α-³²P]dCTP(deoxycytidine 5′-triphosphate, tetra (triethylammonium) salt, [α-³²P])(NEN-Dupont, Boston, Mass.) using the BRL Nick Translation System (BRLLife Technologies, Inc., Gaithersburg, MD), following the supplier'sinstructions. A typical reaction was performed in 0.05 ml volume. Afteraddition of 5 μA Stop buffer, the labeled DNA was separated fromunincorporated nucleotides using a G-25 Sephadex Quick Spin T Column(Boehringer Mannheim) following the supplier's instructions.

Approximately 1800 cosmid clones were screened by colony hybridization.Ten clones were identified that hybridized strongly to the Sacc.erythraea KS probe. E. coli colonies containing cosmid DNA were grown inLB liquid medium and cosmid DNA was isolated from each culture in theAutoGen 540™ automated nucleic acid isolation instrument using Cycle 3(equipment software) according to manufacturer's instructions.Restriction endonuclease mapping and Southern blot hybridizationanalyses revealed that five of the clones contained overlappingchromosomal regions. An S. avermitilis genomic BamHI restriction map ofthe five cosmids (i.e., pSE65, pSE66, pSE67, pSE68, pSE69) wasconstructed by analysis of overlapping cosmids and hybridizations (FIG.4).

6.8. Identification of Regulatory Gene ORFs

The following methods were used to subclone fragments derived from thepSE68 clone. pSE68 (5 μg) was digested with Xbal and EcoRI. The reactionmixture was loaded on a 0.8% SeaPlaque™ GTG agarose gel (FMCBioProducts), a ˜19 Kb Xbal/EcoRI fragment was excised from the gelafter electrophoresis, and the DNA was recovered from the gel usingGELase™ (Epicentre Technologies) using the Fast Protocol. Approximately5 μg of pGEM7Zf(+) (Promega) was digested with Xbal and EcoRI. About 0.5μg of the 19 Kb EcoRI/Xbal fragment and 0.5 μg of digested pGEM7Zf(+)were mixed together and incubated overnight with 1 unit of ligase (NewEngland Biolabs, Inc., Beverly, Mass.) at 15° C. in a total volume of 20μl according to supplier's instructions. After incubation, 5 μl of theligation mixture was incubated at 70° C. for 10 min, cooled to rm temp,and used to transform competent E. coli DH5α cells (BRL) according tomanufacturer's instructions. Plasmid DNA was isolated fromampicillin-resistant transformants and the presence of the ˜19 KbXbal/EcoRI insert was confirmed by restriction analysis. This plasmidwas designated as pSE200.

pSE200 was further modified by Exonuclease III digestion using theErase-a-Base System (Promega) following manufacturer's instructions.Five μg of pSE200 were digested with ClaI. ClaI generates 5′ protrusionswhich were protected from exonuclease digestion by being filled in withalpha-phosphorothioate deoxyribonucleotides according to manufacturer'sinstructions. pSE200 was then digested with EcoRI and aliquots weredigested with SI nuclease for varying times ranging from 30 seconds to12 min. Si nuclease-treated samples of pSE200 were ligated overnight andtransformed into E. coli HB101 (BRL) competent cells followingmanufacturers instructions. Plasmid DNA was isolated fromampicillin-resistant transformants and the size of the insert DNA wasdetermined by restriction enzyme analysis. One isolate was identifiedthat contained a ˜5.9 Kb insert, and this isolate was designated aspSE201 (FIG. 2A) and deposited with the ATCC (Accession No. 203182). Asecond isolate was identified that contained a 8.8 Kb insert, and thisisolate was designated as pSE210 (FIG. 2B).

Approximately 10 μg of pSE201 were isolated using a plasmid DNAisolation kit (Qiagen, Valencia, Calif.) following manufacturer'sinstructions. This DNA was sequenced using an ABI 373A Automated DNASequencer (Perkin Elmer, Foster City, Calif.). Sequence data wasassembled and edited using Genetic Computer Group programs (GCG,Madison, Wis.). The DNA sequence of the ORFs of the regulatory genes,aveR1 and aveR2, are presented identically in both SEQ ID NO:1 and SEQID NO:3. The aveR1 ORF is from nt 1112 to nt 2317 of SEQ ID NO:1. TheaveR2 ORF is from nt 2314 to nt 3021 of SEQ ID NO:1.

A comparison of the amino acid sequence deduced from the aveR1 ORF of S.avermitilis (SEQ ID NO:2) shows 32% identity to the deduced absA1 geneproduct of S coelicolor (SEQ ID NO:5) (FIG. 1A) (Brian et al., 1996, J.Bacteriology 178:3221-3231). A comparison of the amino acid sequencededuced from the aveR2 ORF of S. avermitilis (SEQ ID NO:4) shows 45%identity to the deduced absA2 gene product of S coelicolor (SEQ ID NO:6)(FIG. 1B).

6.9. Construction and Use of Gene Replacement Vectors

A general description of techniques for introducing mutations intoStreptomyces genes is provided by Kieser and Hopwood, 1991, Meth. Enzym.204:430458. A more detailed description is provided by Anzai et al.1998, J. Antibiot. XLI(2):226-233; Stutzman-Engwall et al., 1992, J.Bacteriol. 174(1):144-154; and Oh and Chater, 1997, J. Bact.179:122-127. These publications are incorporated herein by reference.

6.9.1. Inactivation of Both aveR1 and aveR2 Genes

Both the aveR1 and aveR2 genes were inactivated by replacing a 988 bpBg/II/StuI fragment in pSE210 (FIG. 2B) with the erythromycin resistancegene (ermE) from Saccharopolyspora erythraea, as follows. Approximately5 μg of pSE210 was digested with BglII and StuI to release a ˜10.8 Kbfragment. The BglII end was filled in by incubating the DNA with 100 μMfinal concentration of dNTPs in 1×Klenow buffer and 1U Klenow enzyme(Boehringer Mannheim) for 30 min at 37° C. The ˜10.8 Kb fragment waspurified from an agarose gel and incubated in 1×alkaline phosphatasebuffer and 1 U alkaline phosphatase for 1 hr at 50° C. todephosphorylate the ends. After incubation, the dephosphorylatedfragment was purified by phenol/chloroform extraction as described inSection 6.3 and resuspended in TE buffer. The ermE gene was isolatedfrom pIJ4026 (provided by the John Innes Institute, Norwich, U.K.; seealso Bibb et al., 1985, Gene 41:357-368) by digestion with BglII, andthe BglII ends were filled in by incubating the ermE fragment with 100JIM final concentration of dNTPs in 1×Klenow buffer and 1 U Klenowenzyme for 30 min at 37° C. The ˜1.7 Kb ermE fragment was purified froman agarose gel and 0.5 pg mixed with 0.5 jig of the ˜10.8 Kb fragmentand ligated. The ligation mixture was used to transform competent E.coli HB101 cells (BRL) following manufacturer's instructions. PlasmidDNA was isolated from ampicillin-resistant transformants and thepresence of the ermE insert was confirmed by restriction analysis. Thisplasmid, which was designated as pSE214 (FIG. 3A), was transformed intoE. coli DM1 (BRL), and plasmid DNA was isolated fromampicillin-resistant transformants. The insertion of the ermE gene inplace of the 988 bp BglII/StuI fragment removes 752 bp from the aveR1gene and 232 bp from the aveR2 gene.

pSE214, which will not replicate autonomously in S. avermitilis, wasused as a gene replacement vector for integrative gene replacement asfollows. Eight μl of pSE214 DNA (1 pg) was denatured by adding 2 μl of 1M NaOH, incubating for 10 min at 37° C., then rapidly chilling on ice,followed by adding 2 μl of 1 M HCl, according to the procedure of Oh andChater, 1997, above. S. avermitilis protoplasts were transformed withthe denatured pSE214. Transformants were regenerated under selectiveconditions requiring expression of the erythromycin gene so as to induceintegrative gene recombination of the plasmid into the host cellchromosome. Since the plasmid does not replicate autonomously,erythromycin-resistant-transformants should have the plasmid sequencesintegrated into the chromosome by either a single homologousrecombination event between one of the two DNA segments that flank theermE gene and its homologous counterpart in the chromosome, which willresult in the integration of the entire pSE214 vector, or a doublecross-over where a second recombination event occurs between the secondDNA segment that flanks the mutation and its homologous counterpart inthe chromosome, which will result in the exchange of aveR1/aveR2(inactivated) sequences from the plasmid into the chromosome and theconcomitant loss of the wild-type allele and the vector sequences fromthe chromosome. Erythromycin-resistant transformants were isolated andscreened by PCR for the presence of the vector backbone. Alltransformants were missing the vector backbone, suggesting that a doublecross-over event had taken place and the chromosomal aveR1/aveR2sequences had been replaced by the aveR1/aveR2 deleted sequences.Erythromycin-resistant transformants were analyzed by HPLC analysis offermentation products. S. avermitilis strains containing an aveR1/aveR2deletion produced an average of 3.4 times as much total avermectins asthe control strain (TABLE 1).

6.9.2. Inactivation of the aveR2 Gene

The aveR2 gene was inactivated by inserting the ermE gene into a BglIIsite in pSE210 (FIG. 2B), and using the resulting plasmid as a genereplacement vector in S. avermitilis, as follows. Approximately 5 μg ofpSE210 were digested with BglII. The linearized pSE210 was thenincubated in 1×alkaline phosphatase buffer and 1U alkaline phosphataseto dephosphorylate the ends for 1 hr at 50° C. The ermE gene wasisolated from plJ4026 by digestion with BglII, followed byelectrophoresis, and the ˜1.7 Kb ermE gene was purified from the gel.About 0.5 jig of linearized pSE210 and 0.5 μg of the ermE fragment weremixed together and ligated. The ligation mixture was transformed intocompetent E. coli HB101 (BRL) following manufacturer's instructions.Plasmid DNA was isolated from ampicillin-resistant transformants and thepresence of the ermE insert was confirmed by restriction analysis. Thisplasmid, which was designated as pSE216 (FIG. 3B), was transformed intoE. coli DM1 (BRL) following manufacturer's instructions, and plasmid DNAwas isolated from ampicillin-resistant transformants.

pSE216, which will not replicate autonomously in S. avermitilis, wasused as a gene replacement vector for integrative gene replacement asfollows. Eight μl of pSE216 DNA (1 μg) was denatured by adding 2 μl 1MNaOH, incubating for 10 min at 37° C., then rapidly chilling on ice,followed by adding 2 μl of 1M HCl (Oh and Chater, 1997, above). S.avermitilis protoplasts were then transformed with denatured pSE216.Transformants were generated under selective conditions that requireexpression of the erythromycin gene to induce integrative generecombination of the plasmid into the host cell chromosome. Since theplasmid does not replicate autonomously, erythromycin-resistanttransformants should have the plasmid sequences integrated into thechromosome by either a single homologous recombination event between oneof the two DNA segments that flank the ermE gene and its homologouscounterpart in the chromosome, which will result in the integration ofthe entire pSE216 vector, or a double cross-over where a secondrecombination event occurs between the second DNA segment that flanksthe mutation and its homologous counterpart in the chromosome, whichwill result in the exchange of the aveR2 (inactivated) sequences fromthe plasmid into the chromosome and the concomitant loss of thewild-type allele and the vector sequences from the chromosome.Erythromycin-resistant transformants were isolated and screened by PCRfor the presence of the vector backbone. All transformants were missingthe vector backbone, suggesting that a double cross-over event had takenplace and the chromosomal aveR2 sequences had been replaced by theinactivated aveR2 sequence. Erythromycin-resistant transformants wereanalyzed by HPLC analysis of fermentation products. S. avermitilisstrains containing an inactivated aveR2 gene produced an average of 3.1times as much total avermectins as the control strain (TABLE 1).

TABLE 1 Control aveR1/aveR2 aveR2 Insertion (avg. of 5 Deletion (avg.of6 cultures) (avg. of 4 cultures) cultures) Total Relative 1 3.4 3.1 Amt.Of Avermectins

DEPOSIT OF BIOLOGICAL MATERIALS

The following biological material was deposited with the American TypeCulture Collection (ATCC) at 12301 Parklawn Drive, Rockville, Md.,20852, USA, on September 9, 1998, and was assigned the followingaccession numbers:

Plasmid Accession No. plasmid pSE201 203182

All patents, patent applications, and publications cited above areincorporated herein by reference in their entirety.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

6 1 5045 DNA Streptomyces avermitilis CDS (1112)..(2317) aveR1 ORF 1cgagttgctg gtcatcggcg tactcctccc ggcgactccg cccggtactc gaccgcggca 60gcggtcagcc gcatgaacgc ctcttcgaga gacacggtct tccgcgtcac ctcgtgcacc 120gtcacggcgt gcgccgcgac gagttcgccg atccgctccg cggcggcggc caccttcagg 180ctgccgtcgg agcagtcggt gaccgtgatt cccgcgccga ccagcacgtc gcgcagccgc 240cgcggttccg gagtgcgcac ccgcaccccc acgtccgtgt acctgtcgat gaactcgctc 300atgctggtgt ccgcgaggag ccgaccgcgg ccgatgatga cgaggtggtc cacggtgagc 360gccgcctcgc tcatcagatg gctggacacg aagacggtgc gtccctgtgc cgccaggtcc 420cgcatgaggt gccgcagcca caggacgcct tccgggtcga gcccgttgac cggctcgtcg 480agcaccagga cggcggggtc gccgagcagc gccgcggcga ttcccagccg ctgactcatg 540cccagcgaga acgtccccgc ccgccggcgc acggcgctcc gcaggcccgc cagctcgatc 600acctcacgga cgcggcgggg cgggatccgg ttgctgcggg ccagccagcg caagtggttc 660agcgcggttc ggccggggtg caccgccctg gcgtcgagca gtgcccccac cgtccgcagc 720gggtcgcgga gccgctggta gggcgcgccg tcgatgcgta cctcgccggc cgtcgggcgg 780tccaggccca gcatcatgcg catcgcggtg gacttcccgg cgccgttggg gccaaggaat 840ccggtcaccc gaccggtccg tacctggaag gtaagaccct ccacaacggt ggtggtcccg 900tagcgcttgg tcaggtccgt gacttcgatc atgccggtga tggtccgtga cgacaggctc 960ccgccgcgtc ccgctcgggg ctgactgccc cttctccacc cccggttgga gaatgaccgc 1020cacccgcggc cgcgcatcag gctgcaggag gagcggcttt gaccaccgct ggacggaggc 1080ggagcggcgt acgcctggat atggtcgagc g gtg cat gca ggt acc gcg gtg 1132 ValHis Ala Gly Thr Ala Val 1 5 gac ccc gac gac cat ccg atc ctg gcc cgg cgactg agc cgg cgc cga 1180 Asp Pro Asp Asp His Pro Ile Leu Ala Arg Arg LeuSer Arg Arg Arg 10 15 20 ctc atc gcc ctg gac ggc gtg ctc gta ttc gcc tacgca tgc gcg ctg 1228 Leu Ile Ala Leu Asp Gly Val Leu Val Phe Ala Tyr AlaCys Ala Leu 25 30 35 ctg tcg acc ggg ccg aca ggc atc tcg tcg tcg tcc gcgccg ccg ctc 1276 Leu Ser Thr Gly Pro Thr Gly Ile Ser Ser Ser Ser Ala ProPro Leu 40 45 50 55 ccg gcc ccg gtg ccg tgg gag cgg ctc gtg ctc atc gccgcg gcc act 1324 Pro Ala Pro Val Pro Trp Glu Arg Leu Val Leu Ile Ala AlaAla Thr 60 65 70 gcg cct gtc gcc gta cgg cgg atc tgg ccg ttg ccc gtg ttcgcg gtc 1372 Ala Pro Val Ala Val Arg Arg Ile Trp Pro Leu Pro Val Phe AlaVal 75 80 85 gtg ctg gcg gtg acc gcc gtg gcc gtc gtg cgg gac gcg gcg tgggac 1420 Val Leu Ala Val Thr Ala Val Ala Val Val Arg Asp Ala Ala Trp Asp90 95 100 ccg ttc ctg tcg gcg gcg ttc gcc ctc tac acc gtc gcc gtc acggtg 1468 Pro Phe Leu Ser Ala Ala Phe Ala Leu Tyr Thr Val Ala Val Thr Val105 110 115 ccc tcg cgc cac tgg tgg caa cgc tgg tta ccc ggc ctg gcg atcgct 1516 Pro Ser Arg His Trp Trp Gln Arg Trp Leu Pro Gly Leu Ala Ile Ala120 125 130 135 ttg ctg acc gtg gcc ggc ctt gcc gga gca gcg cgt gcc ggcgag gcc 1564 Leu Leu Thr Val Ala Gly Leu Ala Gly Ala Ala Arg Ala Gly GluAla 140 145 150 ttc tgg tgg cgc ggc agc ccc ggt ctg ctg ctg ctc ggc ttcgcc gca 1612 Phe Trp Trp Arg Gly Ser Pro Gly Leu Leu Leu Leu Gly Phe AlaAla 155 160 165 ctg ctc ggc gcc tgg caa ctg gga cgc gcc gcg cgg cag aggcgc gca 1660 Leu Leu Gly Ala Trp Gln Leu Gly Arg Ala Ala Arg Gln Arg ArgAla 170 175 180 ttc gcc gtc cgg gcg gcc gag cag ctc gca caa cgg gcc gtcacg gag 1708 Phe Ala Val Arg Ala Ala Glu Gln Leu Ala Gln Arg Ala Val ThrGlu 185 190 195 gaa cgc ctg cgg ata gcc cgc gaa ctg cat gac gtc gtc acgcac agc 1756 Glu Arg Leu Arg Ile Ala Arg Glu Leu His Asp Val Val Thr HisSer 200 205 210 215 atg ggc ctg atc gcg gtc aag gtc ggc gtc gcc aac cacgtg ttg cac 1804 Met Gly Leu Ile Ala Val Lys Val Gly Val Ala Asn His ValLeu His 220 225 230 atc agg ccg cag gag gcg tac gac gcg ctc cag gtc atcgaa cgc acg 1852 Ile Arg Pro Gln Glu Ala Tyr Asp Ala Leu Gln Val Ile GluArg Thr 235 240 245 agc cgc acc gcg ctg aac gac atg cgc cgg atg ctc ggtgtg ctg cgt 1900 Ser Arg Thr Ala Leu Asn Asp Met Arg Arg Met Leu Gly ValLeu Arg 250 255 260 acg tcc gag ggt gag cgg cag tca gcg gct ctc ggc ccgctg cct ggc 1948 Thr Ser Glu Gly Glu Arg Gln Ser Ala Ala Leu Gly Pro LeuPro Gly 265 270 275 gcc ctt gct ctc cct gac ctc gtc ggg cag gcc ggc gcgcag ctg act 1996 Ala Leu Ala Leu Pro Asp Leu Val Gly Gln Ala Gly Ala GlnLeu Thr 280 285 290 295 atg cgc ggt gtc gag agt ctg ccc gac gga gtc gcgctg gcc gtc tac 2044 Met Arg Gly Val Glu Ser Leu Pro Asp Gly Val Ala LeuAla Val Tyr 300 305 310 cgg atc gtg cag gag gcg ctc acc aat gtc gcc aagcac gcc ggc ccg 2092 Arg Ile Val Gln Glu Ala Leu Thr Asn Val Ala Lys HisAla Gly Pro 315 320 325 gag gcc cgc tgc cgg gtg gcg gtc gat gcg aac ggccac ggc gtc cgg 2140 Glu Ala Arg Cys Arg Val Ala Val Asp Ala Asn Gly HisGly Val Arg 330 335 340 ctc gag ata acc gac gac gga ggc gac cgg agc cccctc gcg ccg aag 2188 Leu Glu Ile Thr Asp Asp Gly Gly Asp Arg Ser Pro LeuAla Pro Lys 345 350 355 ccc ggc ggc cac gga atc gtc ggc atg cgc gaa cgcgtc gcc ctg tac 2236 Pro Gly Gly His Gly Ile Val Gly Met Arg Glu Arg ValAla Leu Tyr 360 365 370 375 ggc ggc acc ttc gcc gcc gga ccg cgt cca gagggc ggc ttc gcg gta 2284 Gly Gly Thr Phe Ala Ala Gly Pro Arg Pro Glu GlyGly Phe Ala Val 380 385 390 cac gcg tcc ctg ccg tac gag gag aac aca tgacccggcccgc cgatccgccc 2337 His Ala Ser Leu Pro Tyr Glu Glu Asn Thr 395400 ggtgccccgg tccgggtcct catcgccgac gaccaggcgc tgctgcgcgg cagcctgcgg2397 gtgctcgtcg acaccgagcc cggcctggtg gccacgtcgg aggcggcgac cggcacggag2457 gcggtgcggc ttgcccggca ggatccgccg gacgtggtcc tgatggacgt gcggatgccc2517 gaaatggatg gcatcgaggc gacccggcag atctgcggtt cccccgagac cgcggacgtc2577 aaagtgctga tcctgacgat gttcgacctg gacgagtacg tctacgccgc gctgcgggcc2637 ggtgccagcg gcttcctgct gaaggacacg ccgcccagcg agttgctcgc ggcggtacgg2697 gtcatcgccg ccggcgaggc gctgctggca ccggccgtga cgcggcgcct gatcgcggag2757 ttcgtccacc gcccggagcc ctcgcgaccg ctgcgtcgca ccctggacgg cgtgaccgag2817 cgcgaacgtg aagtcctcac cctcatcgcc tgcggcctgt ccaacaccga gatcgccgag2877 cggctgtatc tcggcattgc caccgtgaag acccacgtca gccacctgct caccaagctc2937 gccacccgcg atcgcgctca gttggtgatc gtcgcgtacg agagcggcct ggtcacggtg2997 gcgcgaccac cgatcggttc ctgaggggcg ccggcgcaca cggtgcacgg cctgggcggg3057 gccgttcaga atggatcacc cgggtacacg aggcgcagtt cgtcgacatg gctcatgagg3117 tactcaccgg ggcactgggt ggatgccggg gcccgggact gcttcttgcg cggctggtgg3177 ccccagacgc tgctgatgcc gaagcggacg gccaggacgt ccacgaggac gtcgagtgtt3237 gtgagttgct tgggcgtcgg gtggtcgtag cgtgcccact ggttctgcca gcgcggtccg3297 aagtcgccgg tgagcacgat gccgagattg ccggcgttga agagttcagc gtgtgagccc3357 tcgatgccga gtggccgccc ctcgtagatc gtcccggcgc cgtcgatgat gtagtggtaa3417 ccgatgtcgg ccttgtcgtc cgcgaagtgc gcccgctgga tcgtgcgcgg gccctcatgc3477 gtgtacgtga cggggtcggc cgagtggtgg atggtgatcc agcggtagac ggaggccagg3537 ggccggttct cgctgagcgg tacgggactg ccgcggtagg gcggtggcgc gagggggccg3597 gaggcggcct cgtggaaatc ccaggtacgc agcggggggt cgatctgcgg cggggccgcc3657 ccccaggtgg cgcggccgac gacggacacg gtcagcggcc ctcgcggtgt catggcccac3717 aactcgtagt cgccgctcgc cggatgcagg aagcgcgact cgtcccagca ggcggcgacg3777 gggccgtgcg cggtgtcgac cggccgggtg ccgcaggacg tcagcctcag gggagtccgg3837 tgcgggcgct gccccgagac cggcgcgttg aaccggccga tgtcggtgat cacggtggtg3897 cggagttccg acaggtcgta gccgtcgcgg gggcattcga gggagcgcgg cggcggttcc3957 acgaccctga gcgccgcatc gcaccggggg cagacgagaa cgagcacctc gcgggcgacc4017 agctccgtcg tcgtaccggg cgggagccgg tggtggcggg gcagatcgag tggcgtgcgg4077 ccgggccgca gttcggtcac gggcacgggg tcggtggctt cggcggcggg tgccagctcg4137 tggtcggcgc aggcgaccgt ccaggcacgc gtcccggcgt cgggaaccat gagggtgccc4197 agcgcgtccg tcgtggccgc gatcccggaa tgccgtcctc ccgatggcgg gatgagccgt4257 acggtgaatc cggggatcgg gctgccgtcg cggcgcagga tcaccagggc cgtgtccgac4317 ggtggtgaga gttcggcggc cagccccgcc tcgacgaagt gcagcaagcg gtgtgtcagt4377 tgcagtacct cgggagagtc cggcgcgagc atggcctcgg cacggctgcg cacgctctcg4437 aacgcgccgc cgagtgcgaa gcgcaggaag tcgacggcga acgcgacgat ctccccggcg4497 acgaagccga ccgcggcgtc ggcgaagcga ggcgggccga agccaggtgc cagggggagc4557 gccggcgctc cggcactggt cctggtggcg gcgacgaacg cggtgcaacg ccggtccacg4617 gcgccgtcgt agtactcacg cagctgcgcc gccagcgagc ggtgcgggtc gaaggactcg4677 ccgaggttca ccccgtcgat gtcgcccagc agccgcggcg tcgaagcgtg gcgggcgacc4737 cagtggtcca gcgaccgacc gcggtccgcg gccggcaccc cgggcgcgtg gcgggcgcgg4797 acgtacgcgg cgagggcgcg cccgaggtca ccgctccagg tgagggcgag atccgctcga4857 ggggccgggt ccagggggcc gggcgtctgc cggtcggccc cgtcgatgcc ggccagcacc4917 tgcgccaggt cgagccgctc gaagccgtgc tgcacccgca gcagcgcggc cagccgggcg4977 gcccggcggg gcagctccca ggacgagccc ggcgtctggt cgtacggggg gatgttccgc5037 cggttctg 5045 2 401 PRT Streptomyces avermitilis 2 Val His Ala GlyThr Ala Val Asp Pro Asp Asp His Pro Ile Leu Ala 1 5 10 15 Arg Arg LeuSer Arg Arg Arg Leu Ile Ala Leu Asp Gly Val Leu Val 20 25 30 Phe Ala TyrAla Cys Ala Leu Leu Ser Thr Gly Pro Thr Gly Ile Ser 35 40 45 Ser Ser SerAla Pro Pro Leu Pro Ala Pro Val Pro Trp Glu Arg Leu 50 55 60 Val Leu IleAla Ala Ala Thr Ala Pro Val Ala Val Arg Arg Ile Trp 65 70 75 80 Pro LeuPro Val Phe Ala Val Val Leu Ala Val Thr Ala Val Ala Val 85 90 95 Val ArgAsp Ala Ala Trp Asp Pro Phe Leu Ser Ala Ala Phe Ala Leu 100 105 110 TyrThr Val Ala Val Thr Val Pro Ser Arg His Trp Trp Gln Arg Trp 115 120 125Leu Pro Gly Leu Ala Ile Ala Leu Leu Thr Val Ala Gly Leu Ala Gly 130 135140 Ala Ala Arg Ala Gly Glu Ala Phe Trp Trp Arg Gly Ser Pro Gly Leu 145150 155 160 Leu Leu Leu Gly Phe Ala Ala Leu Leu Gly Ala Trp Gln Leu GlyArg 165 170 175 Ala Ala Arg Gln Arg Arg Ala Phe Ala Val Arg Ala Ala GluGln Leu 180 185 190 Ala Gln Arg Ala Val Thr Glu Glu Arg Leu Arg Ile AlaArg Glu Leu 195 200 205 His Asp Val Val Thr His Ser Met Gly Leu Ile AlaVal Lys Val Gly 210 215 220 Val Ala Asn His Val Leu His Ile Arg Pro GlnGlu Ala Tyr Asp Ala 225 230 235 240 Leu Gln Val Ile Glu Arg Thr Ser ArgThr Ala Leu Asn Asp Met Arg 245 250 255 Arg Met Leu Gly Val Leu Arg ThrSer Glu Gly Glu Arg Gln Ser Ala 260 265 270 Ala Leu Gly Pro Leu Pro GlyAla Leu Ala Leu Pro Asp Leu Val Gly 275 280 285 Gln Ala Gly Ala Gln LeuThr Met Arg Gly Val Glu Ser Leu Pro Asp 290 295 300 Gly Val Ala Leu AlaVal Tyr Arg Ile Val Gln Glu Ala Leu Thr Asn 305 310 315 320 Val Ala LysHis Ala Gly Pro Glu Ala Arg Cys Arg Val Ala Val Asp 325 330 335 Ala AsnGly His Gly Val Arg Leu Glu Ile Thr Asp Asp Gly Gly Asp 340 345 350 ArgSer Pro Leu Ala Pro Lys Pro Gly Gly His Gly Ile Val Gly Met 355 360 365Arg Glu Arg Val Ala Leu Tyr Gly Gly Thr Phe Ala Ala Gly Pro Arg 370 375380 Pro Glu Gly Gly Phe Ala Val His Ala Ser Leu Pro Tyr Glu Glu Asn 385390 395 400 Thr 3 5045 DNA Streptomyces avermitilis CDS (2314)..(3021)aveR2 ORF 3 cgagttgctg gtcatcggcg tactcctccc ggcgactccg cccggtactcgaccgcggca 60 gcggtcagcc gcatgaacgc ctcttcgaga gacacggtct tccgcgtcacctcgtgcacc 120 gtcacggcgt gcgccgcgac gagttcgccg atccgctccg cggcggcggccaccttcagg 180 ctgccgtcgg agcagtcggt gaccgtgatt cccgcgccga ccagcacgtcgcgcagccgc 240 cgcggttccg gagtgcgcac ccgcaccccc acgtccgtgt acctgtcgatgaactcgctc 300 atgctggtgt ccgcgaggag ccgaccgcgg ccgatgatga cgaggtggtccacggtgagc 360 gccgcctcgc tcatcagatg gctggacacg aagacggtgc gtccctgtgccgccaggtcc 420 cgcatgaggt gccgcagcca caggacgcct tccgggtcga gcccgttgaccggctcgtcg 480 agcaccagga cggcggggtc gccgagcagc gccgcggcga ttcccagccgctgactcatg 540 cccagcgaga acgtccccgc ccgccggcgc acggcgctcc gcaggcccgccagctcgatc 600 acctcacgga cgcggcgggg cgggatccgg ttgctgcggg ccagccagcgcaagtggttc 660 agcgcggttc ggccggggtg caccgccctg gcgtcgagca gtgcccccaccgtccgcagc 720 gggtcgcgga gccgctggta gggcgcgccg tcgatgcgta cctcgccggccgtcgggcgg 780 tccaggccca gcatcatgcg catcgcggtg gacttcccgg cgccgttggggccaaggaat 840 ccggtcaccc gaccggtccg tacctggaag gtaagaccct ccacaacggtggtggtcccg 900 tagcgcttgg tcaggtccgt gacttcgatc atgccggtga tggtccgtgacgacaggctc 960 ccgccgcgtc ccgctcgggg ctgactgccc cttctccacc cccggttggagaatgaccgc 1020 cacccgcggc cgcgcatcag gctgcaggag gagcggcttt gaccaccgctggacggaggc 1080 ggagcggcgt acgcctggat atggtcgagc ggtgcatgca ggtaccgcggtggaccccga 1140 cgaccatccg atcctggccc ggcgactgag ccggcgccga ctcatcgccctggacggcgt 1200 gctcgtattc gcctacgcat gcgcgctgct gtcgaccggg ccgacaggcatctcgtcgtc 1260 gtccgcgccg ccgctcccgg ccccggtgcc gtgggagcgg ctcgtgctcatcgccgcggc 1320 cactgcgcct gtcgccgtac ggcggatctg gccgttgccc gtgttcgcggtcgtgctggc 1380 ggtgaccgcc gtggccgtcg tgcgggacgc ggcgtgggac ccgttcctgtcggcggcgtt 1440 cgccctctac accgtcgccg tcacggtgcc ctcgcgccac tggtggcaacgctggttacc 1500 cggcctggcg atcgctttgc tgaccgtggc cggccttgcc ggagcagcgcgtgccggcga 1560 ggccttctgg tggcgcggca gccccggtct gctgctgctc ggcttcgccgcactgctcgg 1620 cgcctggcaa ctgggacgcg ccgcgcggca gaggcgcgca ttcgccgtccgggcggccga 1680 gcagctcgca caacgggccg tcacggagga acgcctgcgg atagcccgcgaactgcatga 1740 cgtcgtcacg cacagcatgg gcctgatcgc ggtcaaggtc ggcgtcgccaaccacgtgtt 1800 gcacatcagg ccgcaggagg cgtacgacgc gctccaggtc atcgaacgcacgagccgcac 1860 cgcgctgaac gacatgcgcc ggatgctcgg tgtgctgcgt acgtccgagggtgagcggca 1920 gtcagcggct ctcggcccgc tgcctggcgc ccttgctctc cctgacctcgtcgggcaggc 1980 cggcgcgcag ctgactatgc gcggtgtcga gagtctgccc gacggagtcgcgctggccgt 2040 ctaccggatc gtgcaggagg cgctcaccaa tgtcgccaag cacgccggcccggaggcccg 2100 ctgccgggtg gcggtcgatg cgaacggcca cggcgtccgg ctcgagataaccgacgacgg 2160 aggcgaccgg agccccctcg cgccgaagcc cggcggccac ggaatcgtcggcatgcgcga 2220 acgcgtcgcc ctgtacggcg gcaccttcgc cgccggaccg cgtccagagggcggcttcgc 2280 ggtacacgcg tccctgccgt acgaggagaa cac atg acc cgg ccc gccgat ccg 2334 Met Thr Arg Pro Ala Asp Pro 1 5 ccc ggt gcc ccg gtc cgg gtcctc atc gcc gac gac cag gcg ctg ctg 2382 Pro Gly Ala Pro Val Arg Val LeuIle Ala Asp Asp Gln Ala Leu Leu 10 15 20 cgc ggc agc ctg cgg gtg ctc gtcgac acc gag ccc ggc ctg gtg gcc 2430 Arg Gly Ser Leu Arg Val Leu Val AspThr Glu Pro Gly Leu Val Ala 25 30 35 acg tcg gag gcg gcg acc ggc acg gaggcg gtg cgg ctt gcc cgg cag 2478 Thr Ser Glu Ala Ala Thr Gly Thr Glu AlaVal Arg Leu Ala Arg Gln 40 45 50 55 gat ccg ccg gac gtg gtc ctg atg gacgtg cgg atg ccc gaa atg gat 2526 Asp Pro Pro Asp Val Val Leu Met Asp ValArg Met Pro Glu Met Asp 60 65 70 ggc atc gag gcg acc cgg cag atc tgc ggttcc ccc gag acc gcg gac 2574 Gly Ile Glu Ala Thr Arg Gln Ile Cys Gly SerPro Glu Thr Ala Asp 75 80 85 gtc aaa gtg ctg atc ctg acg atg ttc gac ctggac gag tac gtc tac 2622 Val Lys Val Leu Ile Leu Thr Met Phe Asp Leu AspGlu Tyr Val Tyr 90 95 100 gcc gcg ctg cgg gcc ggt gcc agc ggc ttc ctgctg aag gac acg ccg 2670 Ala Ala Leu Arg Ala Gly Ala Ser Gly Phe Leu LeuLys Asp Thr Pro 105 110 115 ccc agc gag ttg ctc gcg gcg gta cgg gtc atcgcc gcc ggc gag gcg 2718 Pro Ser Glu Leu Leu Ala Ala Val Arg Val Ile AlaAla Gly Glu Ala 120 125 130 135 ctg ctg gca ccg gcc gtg acg cgg cgc ctgatc gcg gag ttc gtc cac 2766 Leu Leu Ala Pro Ala Val Thr Arg Arg Leu IleAla Glu Phe Val His 140 145 150 cgc ccg gag ccc tcg cga ccg ctg cgt cgcacc ctg gac ggc gtg acc 2814 Arg Pro Glu Pro Ser Arg Pro Leu Arg Arg ThrLeu Asp Gly Val Thr 155 160 165 gag cgc gaa cgt gaa gtc ctc acc ctc atcgcc tgc ggc ctg tcc aac 2862 Glu Arg Glu Arg Glu Val Leu Thr Leu Ile AlaCys Gly Leu Ser Asn 170 175 180 acc gag atc gcc gag cgg ctg tat ctc ggcatt gcc acc gtg aag acc 2910 Thr Glu Ile Ala Glu Arg Leu Tyr Leu Gly IleAla Thr Val Lys Thr 185 190 195 cac gtc agc cac ctg ctc acc aag ctc gccacc cgc gat cgc gct cag 2958 His Val Ser His Leu Leu Thr Lys Leu Ala ThrArg Asp Arg Ala Gln 200 205 210 215 ttg gtg atc gtc gcg tac gag agc ggcctg gtc acg gtg gcg cga cca 3006 Leu Val Ile Val Ala Tyr Glu Ser Gly LeuVal Thr Val Ala Arg Pro 220 225 230 ccg atc ggt tcc tga ggggcgccggcgcacacggt gcacggcctg ggcggggccg 3061 Pro Ile Gly Ser 235 ttcagaatggatcacccggg tacacgaggc gcagttcgtc gacatggctc atgaggtact 3121 caccggggcactgggtggat gccggggccc gggactgctt cttgcgcggc tggtggcccc 3181 agacgctgctgatgccgaag cggacggcca ggacgtccac gaggacgtcg agtgttgtga 3241 gttgcttgggcgtcgggtgg tcgtagcgtg cccactggtt ctgccagcgc ggtccgaagt 3301 cgccggtgagcacgatgccg agattgccgg cgttgaagag ttcagcgtgt gagccctcga 3361 tgccgagtggccgcccctcg tagatcgtcc cggcgccgtc gatgatgtag tggtaaccga 3421 tgtcggccttgtcgtccgcg aagtgcgccc gctggatcgt gcgcgggccc tcatgcgtgt 3481 acgtgacggggtcggccgag tggtggatgg tgatccagcg gtagacggag gccaggggcc 3541 ggttctcgctgagcggtacg ggactgccgc ggtagggcgg tggcgcgagg gggccggagg 3601 cggcctcgtggaaatcccag gtacgcagcg gggggtcgat ctgcggcggg gccgcccccc 3661 aggtggcgcggccgacgacg gacacggtca gcggccctcg cggtgtcatg gcccacaact 3721 cgtagtcgccgctcgccgga tgcaggaagc gcgactcgtc ccagcaggcg gcgacggggc 3781 cgtgcgcggtgtcgaccggc cgggtgccgc aggacgtcag cctcagggga gtccggtgcg 3841 ggcgctgccccgagaccggc gcgttgaacc ggccgatgtc ggtgatcacg gtggtgcgga 3901 gttccgacaggtcgtagccg tcgcgggggc attcgaggga gcgcggcggc ggttccacga 3961 ccctgagcgccgcatcgcac cgggggcaga cgagaacgag cacctcgcgg gcgaccagct 4021 ccgtcgtcgtaccgggcggg agccggtggt ggcggggcag atcgagtggc gtgcggccgg 4081 gccgcagttcggtcacgggc acggggtcgg tggcttcggc ggcgggtgcc agctcgtggt 4141 cggcgcaggcgaccgtccag gcacgcgtcc cggcgtcggg aaccatgagg gtgcccagcg 4201 cgtccgtcgtggccgcgatc ccggaatgcc gtcctcccga tggcgggatg agccgtacgg 4261 tgaatccggggatcgggctg ccgtcgcggc gcaggatcac cagggccgtg tccgacggtg 4321 gtgagagttcggcggccagc cccgcctcga cgaagtgcag caagcggtgt gtcagttgca 4381 gtacctcgggagagtccggc gcgagcatgg cctcggcacg gctgcgcacg ctctcgaacg 4441 cgccgccgagtgcgaagcgc aggaagtcga cggcgaacgc gacgatctcc ccggcgacga 4501 agccgaccgcggcgtcggcg aagcgaggcg ggccgaagcc aggtgccagg gggagcgccg 4561 gcgctccggcactggtcctg gtggcggcga cgaacgcggt gcaacgccgg tccacggcgc 4621 cgtcgtagtactcacgcagc tgcgccgcca gcgagcggtg cgggtcgaag gactcgccga 4681 ggttcaccccgtcgatgtcg cccagcagcc gcggcgtcga agcgtggcgg gcgacccagt 4741 ggtccagcgaccgaccgcgg tccgcggccg gcaccccggg cgcgtggcgg gcgcggacgt 4801 acgcggcgagggcgcgcccg aggtcaccgc tccaggtgag ggcgagatcc gctcgagggg 4861 ccgggtccagggggccgggc gtctgccggt cggccccgtc gatgccggcc agcacctgcg 4921 ccaggtcgagccgctcgaag ccgtgctgca cccgcagcag cgcggccagc cgggcggccc 4981 ggcggggcagctcccaggac gagcccggcg tctggtcgta cggggggatg ttccgccggt 5041 tctg 5045 4235 PRT Streptomyces avermitilis 4 Met Thr Arg Pro Ala Asp Pro Pro GlyAla Pro Val Arg Val Leu Ile 1 5 10 15 Ala Asp Asp Gln Ala Leu Leu ArgGly Ser Leu Arg Val Leu Val Asp 20 25 30 Thr Glu Pro Gly Leu Val Ala ThrSer Glu Ala Ala Thr Gly Thr Glu 35 40 45 Ala Val Arg Leu Ala Arg Gln AspPro Pro Asp Val Val Leu Met Asp 50 55 60 Val Arg Met Pro Glu Met Asp GlyIle Glu Ala Thr Arg Gln Ile Cys 65 70 75 80 Gly Ser Pro Glu Thr Ala AspVal Lys Val Leu Ile Leu Thr Met Phe 85 90 95 Asp Leu Asp Glu Tyr Val TyrAla Ala Leu Arg Ala Gly Ala Ser Gly 100 105 110 Phe Leu Leu Lys Asp ThrPro Pro Ser Glu Leu Leu Ala Ala Val Arg 115 120 125 Val Ile Ala Ala GlyGlu Ala Leu Leu Ala Pro Ala Val Thr Arg Arg 130 135 140 Leu Ile Ala GluPhe Val His Arg Pro Glu Pro Ser Arg Pro Leu Arg 145 150 155 160 Arg ThrLeu Asp Gly Val Thr Glu Arg Glu Arg Glu Val Leu Thr Leu 165 170 175 IleAla Cys Gly Leu Ser Asn Thr Glu Ile Ala Glu Arg Leu Tyr Leu 180 185 190Gly Ile Ala Thr Val Lys Thr His Val Ser His Leu Leu Thr Lys Leu 195 200205 Ala Thr Arg Asp Arg Ala Gln Leu Val Ile Val Ala Tyr Glu Ser Gly 210215 220 Leu Val Thr Val Ala Arg Pro Pro Ile Gly Ser 225 230 235 5 394PRT Streptomyces coelicolor 5 Met His Arg Trp Gln Ala Val Arg Arg ArgIle Glu Ser Leu Val Arg 1 5 10 15 Val Leu Gly Ser Glu Arg Pro Phe ThrArg Arg Ala Asp Leu Val Leu 20 25 30 Leu Leu Val Leu Leu Val Pro Ser AlaPhe Ala Thr Gly Thr Leu Glu 35 40 45 Thr Ala Pro Val Ala Trp Leu Thr AlaCys Leu Leu Ile Ala Ala Ala 50 55 60 Val Val Val Gln Arg Thr Ala Pro LeuLeu Ser Leu Leu Leu Ala Ala 65 70 75 80 Leu Leu Thr Leu Phe Tyr Pro TrpPhe Gly Ala Asn Leu Trp Pro Ser 85 90 95 Met Ala Thr Val Val Leu Ser CysLeu Ala Gly Arg Arg Leu Thr Arg 100 105 110 Leu Trp Pro Ala His Leu ValPhe Leu Cys Val Ala Ala Ala Gly Leu 115 120 125 Leu Leu Val Ala Thr ValGly Gln Gly Lys Asp Trp Leu Ser Leu Leu 130 135 140 Met Thr Glu Phe ValAla Cys Val Leu Pro Trp Trp Ala Gly Asn Trp 145 150 155 160 Trp Ser GlnArg Thr Ala Leu Thr His Ala Gly Trp Glu His Ala Glu 165 170 175 Gln LeuGlu Trp Arg Gln Arg Tyr Ile Ala Asp Gln Ala Arg Met Lys 180 185 190 GluArg Ala Arg Ile Ala Gln Asp Ile His Asp Ser Leu Gly His Glu 195 200 205Leu Ser Val Met Ala Leu Leu Ala Gly Gly Leu Glu Leu Ala Pro Gly 210 215220 Leu Ser Asp Pro His Arg Glu Ser Val Gly Gln Leu Arg Glu Arg Cys 225230 235 240 Thr Met Ala Thr Glu Arg Leu His Glu Val Ile Gly Leu Leu ArgGlu 245 250 255 Asp Pro Asn Pro Ser Leu Thr Pro Ala Asp Glu Ser Val AlaGln Leu 260 265 270 Val Arg Arg Phe Gln Arg Ser Gly Thr Pro Val Arg PheGln Glu Asp 275 280 285 Gly Ala Arg Asp Arg Pro Gly Thr Pro Leu Leu SerAsp Leu Ala Ala 290 295 300 Tyr Arg Val Val Gln Glu Ala Leu Thr Asn AlaAla Lys His Ala Pro 305 310 315 320 Gly Ala Pro Ile Asp Val Arg Val ThrHis Thr Ala Asp Glu Thr Val 325 330 335 Val Ser Val Val Asn Glu Arg ProGlu Arg Gly Gly Ser Val Pro Ala 340 345 350 Ala Gly Ser Gly Ser Gly LeuIle Gly Leu Asp Glu Arg Val Arg Leu 355 360 365 Ala Gly Gly Thr Leu ArgThr Gly Pro Arg Ala Gly Gly Phe Glu Val 370 375 380 Tyr Ala Arg Leu ProArg Gly Ala Ser Ser 385 390 6 222 PRT Streptomyces coelicolor 6 Met IleArg Val Leu Leu Ala Asp Asp Glu Thr Ile Ile Arg Ala Gly 1 5 10 15 ValArg Ser Ile Leu Thr Thr Glu Pro Gly Ile Glu Val Val Ala Glu 20 25 30 AlaSer Asp Gly Arg Glu Ala Val Glu Leu Ala Arg Lys His Arg Pro 35 40 45 AspVal Ala Leu Leu Asp Ile Arg Met Pro Glu Met Asp Gly Leu Thr 50 55 60 AlaAla Gly Glu Met Arg Thr Thr Asn Pro Asp Thr Ala Val Val Val 65 70 75 80Leu Thr Thr Phe Gly Glu Asp Arg Tyr Ile Glu Arg Ala Leu Asp Gln 85 90 95Gly Val Ala Gly Phe Leu Leu Lys Ala Ser Asp Pro Arg Asp Leu Ile 100 105110 Ser Gly Val Arg Ala Val Ala Ser Gly Gly Ser Cys Leu Ser Pro Leu 115120 125 Val Ala Arg Arg Leu Met Thr Glu Leu Arg Arg Ala Pro Ser Pro Arg130 135 140 Ser Glu Val Ser Gly Glu Arg Thr Thr Leu Leu Thr Lys Arg GluGln 145 150 155 160 Glu Val Leu Gly Met Leu Gly Ala Gly Leu Ser Asn AlaGlu Ile Ala 165 170 175 Gln Arg Leu His Leu Val Glu Gly Thr Ile Lys ThrTyr Val Ser Ala 180 185 190 Ile Phe Thr Gln Leu Glu Val Arg Asn Arg ValGln Ala Ala Ile Ile 195 200 205 Ala Tyr Glu Ala Gly Leu Val Lys Asp AlaAsp Leu Asn Arg 210 215 220

What is claimed is:
 1. An isolated polynucleotide molecule, comprising anucleotide sequence encoding the amino acid sequence of SEQ ID NO:2. 2.The isolated polynucleotide molecule of claim 1, comprising thenucleotide sequence of SEQ ID NO:1 from nt 1112 to nt
 2317. 3. Theisolated polynucleotide molecule of claim 2, comprising the nucleotidesequence of SEQ ID NO:1.
 4. An isolated polynucleotide molecule that hasgreater than about 80% sequence identity to the nucleotide sequence ofSEQ ID NO:1 from nt 1112 to nt 2317, that hybridizes under highlystringent conditions to the complement of a polynucleotide moleculecomprising said nucleotide sequence, and that encodes a gene productwhich regulates avermectin production in Streptomyces avermitilis.
 5. Anisolated polynucleotide molecule, comprising a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO:4.
 6. The isolatedpolynucleotide molecule of claim 5, comprising the nucleotide sequenceof SEQ ID NO:3 from nt 2314 to nt
 3021. 7. The isolated polynucleotidemolecule of claim 6, comprising the nucleotide sequence of SEQ ID NO:3.8. An isolated polynucleotide molecule that has greater than about 80%sequence identity to the nucleotide sequence of SEQ ID NO:3 from nt 2314to nt 3021, that hybridizes under highly stringent conditions to thecomplement of a polynucleotide molecule comprising said nucleotidesequence, and that encodes a gene product which regulates avermectinproduction in Streptomyces avermitilis.
 9. An isolated polynucleotidemolecule, comprising a nucleotide sequence encoding both the amino acidsequence of SEQ ID NO:2 and the amino acid sequence of SEQ ID NO:4. 10.The isolated polynucleotide molecule of claim 9, comprising thenucleotide sequence of SEQ ID NO:1 from nt 1112 to nt 2317 and thenucleotide sequence of SEQ ID NO:1 from nt 2314 to nt
 3021. 11. Theisolated polynucleotide molecule of claim 10, comprising the nucleotidesequence of SEQ ID NO:1 from nt 1112 to nt
 3021. 12. The isolatedpolynucleotide molecule of claim 11, comprising the nucleotide sequenceof SEQ ID NO:1.
 13. An isolated polynucleotide molecule that has greaterthan about 80% sequence identity to the nucleotide sequence of SEQ IDNO:1 from nt 1112 to nt 3021 that hybridizes under highly stringentconditions to the complement of a polynucleotide molecule comprisingsaid nucleotide sequence, and that encodes a gene product or productswhich regulate avermectin production in Streptomyces avermitilis.
 14. Anisolated polynucleotide molecule, comprising a nucleotide sequence thatis at least about 200 nucleotides in length selected from the nucleotidesequence of SEQ ID NO:1 from nt 1 to nt 1111 and from nt 3022 to nt5045.
 15. A recombinant vector comprising a polynucleotide moleculecomprising a nucleotide sequence encoding the amino acid sequence of SEQID NO:2 or SEQ ID NO:4, or encoding both the amino acid sequence of SEQID NO:2 and the amino acid sequence of SEQ ID NO:4, wherein thepolynucleotide molecule is in operative association with one or moreregulatory elements.
 16. The recombinant vector of claim 15, furthercomprising a nucleotide sequence encoding a selectable marker or areporter gene product.
 17. The recombinant vector of claim 15, whereinthe amino acid sequence of SEQ ID NO:2 is encoded by the nucleotidesequence of SEQ ID NO:1 from nt 1112 to nt 2317, and the amino acidsequence of SEQ ID NO:4 is encoded by the nucleotide sequence of SEQ IDNO:3 from nt 2314 to nt
 3021. 18. The recombinant vector of claim 17,which is plasmid pSE201 (ATCC 203182).
 19. A transformed host cell,comprising the recombinant vector of claim
 15. 20. A substantiallypurified or isolated polypeptide, comprising the amino acid sequence ofSEQ ID NO:2 or SEQ ID NO:4.
 21. A method of preparing a substantiallypurified or isolated polypeptide comprising the amino acid sequence ofSEQ ID NO:2 or SEQ ID NO:4, comprising culturing the transformed hostcell of claim 19 under conditions conducive to the expression of thepolypeptide, and recovering the expressed polypeptide from the cellculture in substantially purified or isolated form.
 22. A method forincreasing the amount of avermectins produced by cells of Streptomycesavermitilis comprising: (a) obtaining cells of a strain of Streptomycesavermitilis; (b) introducing into the cells of step (a) a polynucleotidemolecule comprising a nucleotide sequence that hybridizes under highlystringent conditions to the complement of the nucleotide sequence of SEQID NO:1 from nt 1112 to nt 2317; and (c) selecting Streptomycesavermitilis cells from step (b) which produce detectably increasedamounts of avermectin compared to the cells of step (a). wherein theintroduction of the polynucleotide molecule results in mutation of anendogenous aveR1 gene.
 23. The method of claim 22, wherein saidnucleotide sequence is at least about 70% identical to the nucleotidesequence of SEQ ID NO: 1 from nt 1 1112 to nt 2317 and hybridizes underhighly stringent conditions to the complement of the nucleotide sequencefrom SEQ ID NO: 1 from nt 1112 to nt
 2317. 24. The method of claim 22,wherein said nucleotide sequence comprises at least about 20% of thenucleotide sequence of SEQ ID NO: 1 from nt 1112 to nt 2317 andhybridizes under highly stringent conditions to the complement of thenucleotide sequence of SEQ ID NO: 1 from nt 1112 to nt
 2317. 25. Amethod for increasing the amount of avermectins produced by cells ofStreptomyces avermitilis comprising: (a) obtaining cells of a strain ofStreptomyces avermitilis; (b) introducing into the cells of step (a) apolynucleotide molecule comprising a nucleotide sequence that hybridizesunder highly stringent conditions to the complement of the nucleotidesequence of SEQ ID NO:3 from nt 2314 to nt 3021; and (c) selectingStreptomyces avermitilis cells from step (b) which produce detectablyincreased amounts of avermectin compared to the cells of step (a),wherein the introduction of the polynucleotide molecule results inmutation of an endogenous aveR2 gene.
 26. The method of claim 25,wherein said nucleotide sequence is at least about 70% identical to thenucleotide sequence of SEQ ID NO:3 from nt 2314 to nt 3021 andhybridizes under highly stringent conditions to the complement of thenucleotide sequence from SEQ ID NO:3 from nt 2314 to nt
 3021. 27. Themethod of claim 25 wherein said nucleotide sequence comprises at leastabout 25% of the nucleotide sequence of SEQ ID NO:3 from nt 2314 to nt3021 and hybridizes under highly stringent conditions to the complementof the nucleotide sequence of SEQ ID NO:3 from nt 2314 to nt
 3021. 28. Amethod for increasing the amount of avermectins produced by cells ofStreptomyces avermitilis comprising: (a) obtaining cells of a strain ofStreptomyces avermitilis; (b) introducing into the cells of step (a) apolynucleotide molecule comprising a nucleotide sequence that hybridizesunder highly stringent conditions to the complement of the nucleotidesequence of SEQ ID NO:1 from nt 1112 to nt 3021; and (c) selectingStreptomyces avermitilis cells from step (b) which produce detectablyincreased amounts of avermectin compared to the cells of step (a).wherein the introduction of the polynucleotide molecule results inmutation of an endogenous aveR1 and/or aveR2 gene.
 29. The method ofclaim 28, wherein said nucleotide sequence is at least about 70%identical to the nucleotide sequence of SEQ ID NO: 1 from nt 1112 to nt3021, and hybridizes under highly stringent conditions to the complementof the nucleotide sequence of SEQ ID NO:1 from nt 1112 to nt
 3021. 30.The method of claim 28, wherein said nucleotide sequence comprises atleast about 10% of the nucleotide sequence of SEQ ID NO:1 from nt 1112to nt 3021, and hybridizes under highly stringent conditions to thecomplement of the nucleotide sequence of SEQ ID NO:1 from nt 1112 to nt3021.
 31. A method for increasing the amount of avermectins produced bycells of Streptomyces avermitilis comprising; (a) obtaining cells of astrain of Streptomyces avermitilis; (b) introducing into the cells ofstep (a) a polynucleotide molecule comprising a nucleotide sequence thathybridizes under highly stringent conditions to the complement of thenucleotide sequence of SEQ ID NO:1; and (c) selecting Streptomycesavermitilis cells from step (b) which produce detectably increasedamounts of avermectin compared to the cells of step (a), wherein theintroduction of the polynucleotide molecule results in mutation of anendogenous aveR1 and/or aveR2 gene.
 32. The method of claim 31, whereinsaid nucleotide sequence comprises at least about 10% of the nucleotidesequence of SEQ ID NO:1, and hybridizes under highly stringentconditions to the complement of the nucleotide sequence of SEQ ID NO:1.33. The method of any one of claims 22-32, wherein said mutation is asubstitution.
 34. The method of any one of claims 22-32, wherein saidmutation is a deletion, or an insertion of a different or heterologousnucleotide sequence.
 35. The method of any one of claims 22-32, whereinsaid mutation is a deletion or insertion resulting in a frameshift ofthe coding sequence of the aveR1 and/or aveR2 genes.
 36. The method ofany one of claims 22-32, wherein said mutation is an insertion of atermination codon within the coding sequence of the aveR1 and/or aveR2genes or a substitution resulting in a termination codon within thecoding sequence of the aveR1 and/or aveR2 genes.